LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji-Yüksek Lisans

Bu koleksiyon için kalıcı URI

http://hdl.handle.net/11527/19401

Gözat

A comparative study on immobilization of Jack Bean urease on different matrices

(Graduate School, 2023) Okuşluk, Abdullah Said ; Kılıç, Abdullahim ; 874308 ; Molecular Biology – Genetics and Biotechnology Programme

Biotechnological applications of urease enzyme prevalently involve biosensor development, wastewater treatment systems, artificial kidney machines, restoration of cultural heritages, beverage industries, bioregenerative long-distance space travel systems and rehabilitation of agricultural fields. However, the integration of free urease to industrial, medical, or agricultural applications results in considerable drawbacks, such as activity loss in process conditions and high cost of isolation and purification for reuse. To overcome these mentioned restrictions and develop new biotechnological products, in this thesis study, we immobilized soluble urease onto the eggshell membrane and inner epidermis of the onion bulb scale. Natural supports for the immobilization of Jack Bean urease are already available as waste products of the food sector, with materials that are biocompatible, biodegradable, non-toxic and low-cost. Further, considering sustainability and feasibility issues, the eggshell and onion membranes were chosen for this approach. The eggshell membrane is essentially made of cross-linked collagens as a flexible protein fiber. Additionally, the inner epidermis of the onion bulb scale is predominantly composed of microfibrillar cellulose. The surface morphology of these natural membranes, as displayed by SEM imaging, provided a suitable support for the adsorption technique. This method is the simplest, undemanding, and economically attractive enzyme immobilization approach, relying on weak electrostatic interactions or physical bonding. For the immobilization of Jack Bean urease, each supportive membrane was initially washed with water, air-dried, and cut into 1 cm2 pieces. Subsequently they were treated with branched polyethyleneimine to generate polycationic surfaces. This adsorbent is generally recognized as an FDA-approved safe substance. The urease activity was detected by measuring the amount of released ammonia, as an indicator of residual urease activity.
Alteration of titanium surfaces using hyaluronic acid coated mesoporous silica nanoparticles for local drug release

(Graduate School, 2021-12-17) Erşan, Yeliz ; Karataş Yazgan, Ayten ; Önder, Sakip ; 521181123 ; Biology-Genetics and Biotechnology

Nosocomial infection is still an important problem for developed and developing countries as it decreases the effectiveness of the treatment as well as increases the healthcare expenses due to the prolonged stays in units. Most of these infections are biomaterial-based and caused by biofilm forming bacteria on biomaterial surfaces. Therefore, systemic drug administration is used to prevent biomaterials associated infections and to increase success of the implantation. Otherwise, revision surgery is generally required. Revision surgery means more pain for the patient, and it does not guarantee that osteointegration between the implant and surrounding tissue will be as strong as the first implementation. Local drug release using drug eluting implant materials suggests a great opportunity to prevent implant associated infections. Functional coatings containing therapeutic agents can be deposited on these surfaces by using different surface coating techniques such as layer by layer deposition (LBL), electrophoretic deposition (EPD), physical/chemical vapor deposition (CVD/PVD) etc. Local drug release from these surfaces does not only prevent implant associated infections, it also prevents the systemic toxicity. Moreover, sustained local drug release is possible with drug eluting materials. In the present study, a functional coating based on hyaluronic acid (HA) and mesoporous silica nanoparticles (MSNs) was proposed to prevent implant associated infections. For this purpose, drug loaded HA coated MSNs were prepared and deposited onto the Ti implants using EPD technique. Mesoporous silica nanoparticles (MSNs) were synthesized by the sol-gel/emulsion method and analysed using different characterization techniques such as transmission electron microscopy (TEM), scanning electron microscopy (SEM) and dynamic light scattering (DLS). After that, MSNs was silanized by using APTES (3- Triethoxysilylpropylamine) to form amino groups (-NH2) on the MSNs surfaces. In order to form HA coatings on MSN surfaces, HA was dissolved in MES buffer, NH2 -MSNs were added into the solution, EDC (N-(3-Dimethylaminopropyl)-N'-ethyl- carbodiimid-hydrochloride) and NHS (N-hydroxysuccinimide). Characterization of silanized (-NH2-MSNs) and HA-coated MSNs (HA-MSNs) were done by using SEM, Zeta Sizer and Fourier Transform Infrared Spectroscopy (FT-IR). SEM micrographs showed that almost homogeneous spherical MSNs ranging from 185 nm to 240 nm were synthesized. DLS analysis was showed that their sizes are ca. 8.7 μm. The cause of the difference was due to the environment MSNs exposed during characterization. SEM analysis is performed on dry nanoparticles while watery environment is used for DLS analysis. TEM analysis gave similar results with SEM for the sizes of the MSNs. Porous structure of synthesized MSNs were shown in this analysis. Moreover, characteristic peaks at 696 cm-1 and 1540 cm-1 that are attributed to –NH2 groups after silanization and peaks at 1639 cm-1 that are attributed –CH groups following the HA coating were determined in FTIR analysis. According to these results MSNs, -NH2-MSNs and HA-MSNs are synthesized and modified successfully. Ti plates were treated mechanically and chemically prior to deposit nanoparticles. Deposition onto the Ti surfaces were carried out using two different procedures. In the first procedure, HA-MSNs were dissolved in ethanol (70%) and HA coated MSNs deposited onto the Ti substrates for 1, 3 and 5 min. at 30V. In the second procedure, different ratios of MSNs and HA solution (MSNs:HA (w/w; 1:0.5, 0.5:1, 1:1)) were prepared and electrodes were then placed in this solution. Same coating parameters were applied for both coating procedures. The surfaces were analysed using SEM, FTIR and X-ray spectroscopy (EDS) to examine the surface morphology and chemical composition of the surfaces following the coating process. Characterization studies showed that both procedure 1 and procedure 2 can be used to obtain homogenous coatings on the surfaces. Neverthless, MSNs:HA should be 1:0.5 because coatings that were deposited using higher HA concentrations detached from the surfaces due to the thick film formation. In addition, SEM and EDS analysis showed that coating thickness can be increased with prolonged deposition time. The coating thicknesses were determined using mechanical profilometer and different thicknesses from 0.48 to 1.9 micrometer. were measured on the surfaces for different coating times. According to results of analysis, thickness was increased while increasing deposition time. Drug loading and release studies were carried out in PBS for free (synthesized MSNs, -NH2-MSNs, HA-MSNs) particles and coated particles (HA-MSNs) that were prepared using two different procedures. Ciprofloxacin as a model drug was used in this study, and it was both loaded into free MSNs and coated surfaces by diffusion. In accordance with the drug loading studies, drug loading efficiency was higher for HA-MSNs (ca. 80 %) when compared to HA free MSNs (ca. %40). Moreover, it was shown that drug release was possible using free MSNs for ca. 15 hours at 37 oC. Moreover, synthesized and silanized MSNs did not show sufficient drug loading and release rate like HA- MSNs. Finally, drug release from the coated Ti surfaces were examined. According to the drug release profiles, it was possible to have 10 hours drug release was determined from the Ti surfaces. In sum, HA-coated MSNs can be used as a functional coating to design a drug eluting Ti implant material and to prevent implant associated infections.
Biochemical characterisation of truncated amylopullulanase from Thermoanaerobacter brockii brockii

(Graduate School, 2022-06-24) Kayrav, Aycan ; Karagüler Gül, Nevin ; 521201105 ; Molecular Biology-Genetics and Biotechnology

The last point reached as a result of the chemical transformation carried out by nature is life. Enzymes are responsible biomolecules for the chemical transformation that makes life possible. People had benefited from the reactions carried out by enzymes to obtain various products in many fields throughout history, from the times when they did not know the existence of these catalysts. Over time, people had understood how great potential these biocatalysts have with the structure and working principles. Since then the characterisation of novel enzymes and the usage area of these enzymes have been expanded day by day. Today, more than 3.000 enzymes are used in biotechnological and industrial fields and it is reported that the size of this enzyme market has reached 6 billion USD in 2021. The enzymes that dominate this market with a share of 30 % are those used in the food and beverage industry and the ones that have the upper hand in this group are the enzymes used in starch processing applications. Starch, the main component of many agricultural products and the primary source of carbohydrates for most people, is the main source of carbon in the world. In addition to starch, oligosaccharides, disaccharides and monosaccharides obtained as a result of hydrolysis are also used in various fields such as food, pharmaceuticals and biofuels. To obtain these hydrolysis products, starch is subjected to a three-step process: gelatinisation, liquefaction and saccharification. The viscous solution which is obtained by hydrolysis of α-1,4- and α-1,6- glycosidic bonds in the starch structure and gelatinisation is achieved by using α-amylase, β-amylase, glucoamylase and pullulanase enzymes during the liquefaction. During all these processes to remain the enzymes stable for a long time and maintain their activity, pH adjustment and the addition of Ca2+ ionsare required . Remove both the formed salt as a result of pH adjustments and excess Ca2+ ions causes additional steps and all of these lead to an increase in the cost of production. To eliminate these steps and perform a one-step liquefaction-saccharification process can be possible with an alternative enzyme. Amylopullulanases (E.C. 3.2.1.1/41) are enzymes capable of hydrolysing both α-1,4- and α-1,6- glycosidic bonds, while those obtained from extremophilic organisms can maintain their activities under harsh industrial conditions. In this study, the amylopullulanase (TbbApu) enzyme belonging to the Thermoanaerobium brockii brockii organism, is one of the strong candidates for starch hydrolysis processes, is examined. Previously, for the recombinant production of TbbApu, the whole apu gene had been obtained by using the primary walking method by our group and the optimisation of expression studies was in progress. As a result of the studies, pET-28 a (+) vector and E. coli BL21 (DE3) were chosen as the appropriate expression vector and and E. coli BL21 (DE3) the appropriate host, respectively. Additionally, the variants TbbApuΔSH3 without SH3 domain and TbbApuΔCBM20 without CBM20 domain were constructed to investigate the effect of SH3 and CBM20 domains. In the scope of this thesis, apart from TbbApuΔSH3 and TbbApuΔCBM20 variants, four additional truncated constructs namely TbbApuΔX25-1-SH3, TbbApuΔX25-2-SH3, TbbApuΔX25-1 and TbbApuΔX25-2-CBM20 were also obtained to disclose the effects of X25 domain. The biochemical characterisation, raw starch binding ability and kinetic studies of TbbApu and its six variants have been accomplished. The function of the X25 domain on substrate binding, activity and stability of the enzyme has been revealed for the first time with this study. The genes belonging to each construct were amplified by PCR with specific forward and reverse primers that have SacI and NotI restriction sites at their 5' ends and using the whole apu gene as a template. As a result of the amplification, tbbApuΔX25-1-SH3, tbbApuΔX25-2-SH3, tbbApuΔX25-1-CBM20 and tbbApuΔX25-2-CBM20 genes with a length of 4065, 3750, 3375 and 3060 base pairs, respectively, were obtained. Then, the obtained genes and the pET-28 a (+) expression vector were cut with SacI and NotI enzymes to form sticky ends and the ligation reactions were set up for the insertion of the genes into the pET-28 a (+) expression vector. Then, transformation was performed using competent E. coli BL21 (DE3) host cells. For the determination of positive colonies from the colonies obtained as a result of the transformation, half of the colonies were inoculated on agar plates containing red pullulan. 1 µM IPTG and 40 µg/mL kanamycin were also included in the agar plate for induction and selection respectively. For the control of detected positive colonies from the PRR plate, the other half of the colonies were inoculated into Luria-Bertani (LB) medium and plasmid isolation was performed from these cells. Then, the isolated plasmids were cut with FastDigest SacI enzyme and linearised to determine the length of the plasmids. By linearisation, tbbApuΔX25-1-SH3 - pET-28 a (+) vector with a length of 9434 base pairs; 9119 base pairs long tbbApuΔX25-2-SH3 -pET-28 a (+) vector, 8744 base pairs tbbApuΔX25-1-CBM20- pET-28 a (+) vector and 8429 base pairs tbbApuΔX25-2-CBM20- pET-28 a (+) vector were obtained from all selected colonies. After the cloning, TbbApu, TbbApuΔSH3, TbbApuΔCBM20, TbbApuΔX25-1-SH3, TbbApuΔX25-2-SH3, TbbApuΔX25-1-CBM20 and TbbApuΔX25-2-CBM20 were over-expressed using magic media. To purify these recombinant enzymes, purification was achieved in three steps by using metal affinity chromatography, ion exchange chromatography and heat purification respectively. Then, the pullulanase and α-amylase activities of TbbApu and its six variants were checked by the red pullulan and starch-containing polyacrylamide gel. The biochemical characterisation of the enzymes was completed. As a result of the studies, the optimum reaction temperature for pullulanase activities was determined as 70 °C for TbbApu, TbbApuΔSH3 and TbbApuΔCBM20, 75 °C for TbbApuΔX25-1-SH3, TbbApuΔX25-2-SH3 and TbbApuΔX25-1-CBM20 and also 80 °C for TbbApuΔX25-2-CBM20 domain. The optimum reaction temperature for α-amylase activities was specified as 75 °C for TbbApu, TbbApuΔSH3 and TbbApuΔCBM20, and 80 °C for TbbApuΔX25-1-SH3, TbbApuΔX25-2-SH3, TbbApuΔX25-1-CBM20 and TbbApuΔX25-2-CBM20. The pure variants were incubated for 24 hours at variable temperatures to examine the effect of temperature on the stability of enzymes. It was observed that the most stable temperature of 60 °C for TbbApu, TbbApuΔSH3, TbbApuΔCBM20, TbbApuΔX25-1-SH3 and TbbApuΔX25-2-SH3, 50 °C for TbbApuΔX25-1-CBM20 and 40 °C for TbbApuΔX25-2-CBM20. Optimum pH values for both activities were found to be 6.5 for TbbApu, TbbApuΔX25-1-SH3 and TbbApuΔX25-1-CBM20, 6 for TbbApuΔSH3, TbbApuΔCBM20 and TbbApuΔX25-2-SH3, and 7 for TbbApuΔX25-2-CBM20. It has been determined that the enzymes reached 80 % of their α-amylase activities with pH 5 and protected it up to pH 8. When the effects of different pH values on the stability of the enzymes were examined, the most stable pH according to pullulanase activities was found as pH 4 for TbbApu, TbbApuΔSH3, TbbApuΔCBM20 and TbbApuΔX25-2-CBM20, pH 6 for TbbApuΔX25-2-SH3, pH 6.5 for TbbApuΔX25-1-SH3 and TbbApuΔX25-1-CBM20 after 24 hours of incubation. According to their α-amylase activities, the most stable pH was found as pH 3 for TbbApu, TbbApuΔSH3, TbbApuΔCBM20, pH 6 for TbbApuΔX25-2-SH3, TbbApuΔX25-1-CBM20 and TbbApuΔX25-2-CBM20, pH 7 for TbbApuΔX25-1-SH3. In addition, it is represented that TbbApu and its variants can maintain their stability between pH 3 and 8 and TbbApu can be used in starch hydrolysis processes without pH adjustment. When the effect of metal ions was examined, while Mn2+ and Co2+ ions increased both pullulanase and α-amylase activities of TbbApu and its variants whereas, Mg2+, Zn2+ and Al3+ ions decreased both activities of all enzymes except pullulanase activity of TbbApuΔCBM20 variant. Although α-amylase enzymes used in starch processing are Ca2+ ion-dependent, TbbApu and its variants are not dependent on Ca2+ ion for activity and stability, making the enzyme and its variants a strong candidate for starch hydrolsis process. Also, in the presence of 20 % and 50 % hexane and acetone, with some exceptions, both activities of TbbApu and its variants were increased and 20 % and 50 % butanol, DMF and DMSO presence strongly inhibited both activities of the enzymes. When the effects of organic solvents on the stability of enzymes were examined, it was observed that, with some exceptions, the stability of the enzymes increased in the presence of 20 % and 50 % acetone and hexane, compared to both pullulanase and α-amylase activities, after 24 hours of incubation. In the case of inhibitors and detergents, it was observed that inhibitors moderately inhibited pullulanase and α-amylase activities of TbbApu and its variants with some exceptions, whereas all inhibitors increased the pullulanase activity of TbbApuΔCBM20 and nonionic and anionic enzymes inhibited both activities moderately with. However, the activities of all enzymes were strongly inhibited in the presence of CTAB, which is a cationic detergent. Then, the kinetic parameters of the enzymes were elucidated. The results imply that, truncation SH3 domain improves both α-amylase and pullanase activities of the enzyme. However, truncation of CBM20 and X25 domains from TbbApu caused to loss of affinity and specificity of the enzyme to soluble starch and to shift in the specificity of the enzyme to pullulan. The removal of the SH3 domain and also CBM20 domain, the carbohydrate-binding module in the enzymes did not have any effect on the raw starch binding capacity of TbbApu. The removal of the SH3 domain and also CBM20 domain, the carbohydrate binding module in the enzymes did not have any effect on the raw starch binding capacity of TbbApu. However, sequential removal of X25 and SH3 domains resulted in 27.3 % and 58.4 % reduction in raw starch binding ability, while removal of CBM20 and X25 domains resulted in a 90 % decrease in raw starch binding ability. Thus, it was revealed that the X25 domain is involved in binding raw starch. The results of TLC analysis represented that TbbApu and its variants released maltotriose and maltose as a result of pullulan hydrolysis and maltotriose starch hydrolysis, respectively. The results of TLC analysis represented that TbbApu and its variants released maltotriose and maltose in pullulan hydrolysis and maltotriose in starch hydrolysis, respectively.
Characterization of BAG-1S/C-raf interaction targeting peptide

(Graduate School, 2022-07-25) Çebi, Ecenur ; Doğanay Dinler, Gizem ; 521201110 ; Molecular Biology-Genetics and Biotechnology

ERK/MAPK cascade is one of the most important cellular pathways, which regulates many distinct physiological functions such as cell proliferation, migration, differentiation, and apoptosis. Since it has vital roles in many cellular functions, dysregulation of its members usually results in uncontrolled proliferation and development of cancer cells such as breast cancer. Ras and Raf serine/threonine protein kinases are mostly deregulated components of this pathway and mutations in both proteins have been identified approximately one-third of all human cancer types. Although there are several inhibitors against oncoproteins, because of the improving drug resistance, there is a need for the development of new drugs. One of the emerging interest is to target protein-protein interactions (PPIs) related with cancers. Human Raf kinase family has three isoforms: A-Raf, B-Raf, and C-Raf. C-Raf and B-Raf are the important members of ERK pathway. Although B-Raf is dysregulated mostly, C-Raf has ability to suppress apoptosis and hence, both of the Raf kinase is vital for the cancer progression. The Raf kinases are regulated by several phosphorylation events and also, interactions with some proteins like BAG-1. Bcl-2 associated athanogene 1 (BAG-1) which is an anti-apoptotic co-chaperone protein is usually overexpressed in several cancer types making it possible oncoprotein. Moreover, its known that the interaction between BAG-1 and C-Raf promotes the C-Raf activation and stabilization. Therefore, targeting this interaction with small molecules and/or peptides would be effective therapeutics against different cancer types. In our previous studies, the interaction surface between BAG-1S and C-Raf was determined and a peptidomimetic which coded as Pep3 was designed against the BAG-1S from natural sequence of C-Raf. The aim of this study was to characterize the interaction between BAG-1S and Pep3 and also, the interaction between BAG-1S and TPep3 which is cell penetrating form of Pep3. Firstly, His-tagged BAG-1S proteins were produced in mammalian and bacterial cells. Then, BAG-1S proteins were purified by Ni-NTA affinity purification in two steps. After first step, His-tag was cleaved with TEV protease and tagless proteins were eluted as flow-through in second step while the impurities and TEV proteases were remained bound to resin. Then, the purities of both proteins (produced in mammalian and bacterial) were calculated as >80% by SDS-PAGE. Secondary structure analysis of proteins was performed with circular dichroism and both of the proteins has been folded and showed primarily ɑ-helix characteristics. After BAG-1S proteins were characterized. Pep3 and TPep3 characterization were performed by mass spectrometry and circular dichroism analysis. Pep3 and TPep3 has been synthesized by Fmoc-based solid phase peptide synthesis and they were dissolved in 20 mM AMBIC and water respectively since the Pep3 shows hydrophobic characteristics. Molecular weights of peptides were measured as 2.066 kDa for Pep3 and 3.653 kDa for TPep3 which were compatible with the theoretical calculations. Both of the peptides showed β-sheet characteristics approximately 30% combined with random coils. The interaction between BAG-1S and peptides were confirmed by crosslinking reaction with medium length crosslinker DSS (disuccinimidyl suberate). BAG-1S proteins were incubated with peptides separately to form interaction. After they formed interaction, the crosslink reaction was performed with 50-fold molar excess of DSS. The reactions were analyzed by immunoblotting. BAG-1S was shifted 2 kDa when interacts with Pep3 and 4 kDa with TPep3 comparing to the BAG-1S only reaction. These results were confirmed the interaction of BAG-1S with two peptides. The binding kinetics of BAG-1S and Pep3 was measured by Surface Plasmon Resonance (SPR) with multi-cycle kinetics method. BAG-1S protein was captured on Protein G chip with Anti-BAG-1 and different concentrations of Pep3 was injected. Binding kinetics was calculated as 68.56 nM by 1:1 binding model showing that Pep3 binds BAG-1S with high affinity. The effects of TPep3 on MAPK pathway and cell viability of MCF-7 breast cancer cells were analysed. MCF-7 cells were treated with different concentrations (0 uM to 50 uM) of TPep3 and total proteins were analysed with immunoblotting. C-Raf and p-C-RAf (S338) levels were decreased with the increasing concentration of TPep3. As a result of p-C-Raf inactivation, p-MEK levels were also decreased which showed the decrease in ERK pathway. Moreover, B-Raf and p-B-Raf (S446) levels were decreased. So, it can be said that peptide does not only affect the activitation of C-Raf but also B-Raf. To see the effects of peptide on other cell survival pathways, Akt and p-Akt (S473) levels were analysed and it was seen that their levels remained unchanged even with the highest peptide concentration. Lastly, the IC50 value was calculated as approximately 18 uM from the cell viability MTT assay. Together with all the results, TPep3 and Pep3 peptides have potential to be a promising therapeutics for cancer types relying on ERK pathway since the activity of pathway was decreased with the peptide treatment.
Chronological lifespan analysis of stress-resistant yeasts

(Graduate School, 2024-06-28) Akaydın, Aslı Nur ; Çakar, Zeynep Petek ; 521211101 ; Molecular Biology - Genetics and Biotechnology

Saccharomyces cerevisiae has an important place in human life with its wide usage in various processes like fermentation, brewing, bread and wine production from the oldest times in history. Since its genome sequencing in 1996, it has become one of the most well-known and studied model organisms in many different areas of biology such as cell biology, biotechnology, cancer and aging research. Compared to other model organisms, its ease in genetic manipulation and cultivation conditions made it a convenient host for the production of heterologous proteins and economically valuable products. Yeast shares 30% of homology with many human genes, thus it is a convenient platform to study eukaryotic cell metabolism along with disease models. Aging is a common process all living organisms share and involves numerous metabolic and physiological changes that usually result from accumulated damage and deterioration. Despite the developed technology and improved living conditions through every passage of human life, it is estimated that the aging population will cover one-fifth of the whole population of the world by end of the century and age related diseases will cause a socioeconomic burden to governments. Studying aging in humans is complicated because of the long lifespan and economic and ethical concerns. Although a wide range of organisms share similar aging patterns, using yeast provides a convenient and accurate eukaryotic model with a shorter lifespan and easier growth capability. There are two main approaches to studying aging in yeast: chronological lifespan (CLS) and replicative lifespan (RLS). CLS seeks to analyze the lifespan of undivided yeast cells after they enter the stationary phase, mostly caused by decreased nutrients or toxic metabolite accumulation. RLS defines the number of cell divisions a cell undergoes before its death. CLS analysis is particularly useful for analyzing the response and survival of the cell against certain stress factors and modeling G0 cells that arrest their cell cycle. Metabolic engineering is an effective biotechnological approach for improving metabolic processes and product formation of the organism by altering the existing mechanisms or introducing new ones through the usage of recombinant DNA technology. In classical metabolic engineering, information on metabolic, genetic and physiological data of the strain is gathered, then the manipulations on relevant factors are employed to obtain the desired phenotype. Yet in the inverse metabolic engineering approach, for example when evolutionary engineering is employed, the desired phenotype is achieved using laboratory-based evolutionary settings. In this strategy, yeast strains can become resistant to certain stress factors or produce desired molecules throughout the increased stress treatment during culture. After obtaining the desired phenotype, yeast strains are examined by genomic or transcriptomic analyses to further determine the molecular changes in the genome or transcriptome. With more advanced xix technologies such as CRISPR-Cas9, altered genetic traits can be transferred to wild type strains to evoke the same resistant phenotype. In this study, previously obtained stress-resistant S. cerevisiae strains were analyzed for their CLS and viability performances. Stress factors selected for this purpose were antimycin, boron and freeze-thaw stresses which can affect the production efficiency or viability of yeast strains. In parallel with the general evolutionary engineering strategy, strains were obtained by increasing the stress levels gradually, which is the concentration of the compound in the case of antimycin and boron, and repeat numbers in the case of freeze-thaw stress, in selection cultures until the resistant population is achieved. It was shown previously that the evolved strains could become cross resistant to other stress types or their longevity can be affected by the process. The aim of this study was to determine which type of stress resistances can extend or shorten the CLS of the yeast thus affecting the lifespan of the industrial and laboratory yeast strains. For this aim, both quantitative and semi-quantitative CLS analyses were carried out. In the semi-quantitative CLS experiment, OD600 values of the yeast strains were set to 6 before they were spotted onto agar plates every 2nd day with serial dilutions. The longevity of the resistant strains was compared with their control strains visually, based on the growth on the plates. According to the results, P8 which is the freeze-thaw stress-resistant, industrial polyploid strain had a longer CLS than its industrial reference strain, whereas the antimycin and boron-resistant strains did not have a longer CLS than their reference strain. In the second part of the study, quantitative CLS analysis was done by spreading the long-lived industrial P8 strain along with its industrial reference strain R625 and the laboratory reference strain 905 to agar plates. The longevity was measured by counting colony-forming units (CFUs). The experiment was repeated until the viability of the cultures was reduced to 0.0001% from day 0 of the experiment where the viability was accepted as 100%. In the second part of the study, further validating the results from the semi-quantitative analysis, P8 exhibited longer CLS compared to its industrial reference strain and could live until the 10th day of the experiment. Among the various stress-resistant strains tested in this study, only the freeze-thaw stress-resistant, industrial P8 strain was found to have a longer CLS. However, the antimycin and boron-resistant yeast strains did not show a longer CLS compared to their laboratory reference strain. Since the freeze-thaw response was previously associated with oxidative stress response and nutrient metabolism alterations, the longer CLS of the freeze-thaw stress-resistant industrial strain can be related to changes in respective pathways that originated from the evolutionary engineering process. In the scope of the research done for this study, despite being studied in other organisms, the effect of boron resistance on longevity was studied in yeast for the first time. Similarly, antimycin resistance was examined for its effect on longevity in yeast for the first time, as well. Further studies to analyze genomic and transcriptomic changes that occurred by the acquired resistance can be performed and these changes can be transferred to wild-type or reference strains to assess the viability and CLS profiles.
Comparative whole genome sequencing and bioinformatic analysis of afreeze-thaw stress-resistant, industrial Saccharomyces cerevisiae strain

(Graduate School, 2022) Şimşek, Burcu Tuğba ; Çakar, Zeynep Petek ; 737560 ; Molecular Biology - Genetics and Biotechnology Programme

Yeasts have been around for thousands of years; they have benefited people in many fields such as science, medicine, food and agriculture. In particular, Saccharomyces cerevisiae is used in multi-enzyme pathways for the expression of protein biocatalysts and to synthesize chemicals and small molecular weight compounds important for medicine and nutrition. Due to these advances, S. cerevisiae is currently the primary model organism for the study of eukaryotic biology and human diseases. S. cerevisiae is a unicellular eukaryote. It has 16 chromosomes with subcellular organelles containing and these organelles commonly found in eukaryotes. S. cerevisiae has a classical eukaryotic cell cycle (including G1, S, G2, and M). Different strains of S. cerevisiae have been established to fill the gaps and requirements in genetic, biochemistry and physiology research. The CEN.PK family is frequently used in industrial biotechnology research, while the BY strain family derived from the S288c strain is mainly used in genetic studies. Yeast contains a large number of orthologous genes in the human genome. By examining the expression of some genes in yeast, the mechanism in more complex eukaryotes can be understood. S. cerevisiae has highly developed homologous recombination and contributes to the basic knockout operation of genes. Furthermore, S. cerevisiae is an important model for understanding the role of stress response genes in living organisms. S. cerevisiae cells can experience different environmental stress conditions such as metal toxicity, heat or cold shock during growth, essential nutrient limitations, hyperosmotic or hypoosmotic pressure, and ethanol toxicity. To overcome these stress conditions, S. cerevisiae cells have been developed to detect stress signals and respond to these signals through general or specific stress response and protection programs. Cryopreservation is a long-term storage method of various living cells, and the freeze-thaw tensile strength is important in cryopreservation. However, this method includes freezing and thawing processes that cause fatal damage to cells. Under freeze-thaw stress conditions, cells are exposed to more than one type of stress. These are; cold during freezing, dehydration, osmotic, ice crystal formation and oxidative stress during thawing. Therefore, it is important to obtain freeze-thaw tolerant organisms and to examine all freeze-thaw tolerance mechanisms. Yeasts are organisms that have a high survival rate when rapidly frozen at -80 °C. However, it is usually applied to commercial products at -20 °C and is highly damaging to cells, predominantly lethal to cells. Applications of freeze-thaw stress in S. cerevisiae are concerned with inducing this cross-resistance to overcome the effects of freeze-thaw stress. Additional mechanisms at gene expression levels are thought to be triggered and maintained during freeze-thaw exposure to achieve multiple stress tolerances and freeze-thaw stress tolerances. Metabolic engineering; it is defined as enhanced production of metabolites and cellular activities. It is done with through manipulation of the enzymatic, transport and regulatory functions of the cell by modifications of cellular networks including metabolic, gene regulatory and signaling networks using recombinant DNA technology. Metabolic engineering strategies can be divided into two groups as rational engineering and inverse metabolic engineering. Evolutionary engineering is a common strategy used in biological research to achieve the desired phenotype by improving its properties such as high environmental tolerance and improvement of product yield. Evolutionary engineering differs from metabolic engineering in that it is based on random methods; genetic modifications are not directed. Ploidy is the number of complete sets of chromosomes in a cell, which means the number of possible alleles for autosomal and pseudoautosomal genes. Many eukaryotic creatures have two sets of chromosomes (diploid) or more than two sets of chromosomes (polyploid). During the evolution of plants, animals, and fungi, ancient whole-genome duplication (WGD) or hybridization events frequently result in diploid and polyploid conditions. Increased chromosomal sets, development, cellular stress, disease, and evolution all cause polyploidy. Yeasts, which belong to the kingdom of fungi, can exist in both haploid and diploid forms. Polyploid yeasts, on the other hand, are widespread. Allopolyploid cells are formed when two or more cells from closely related but not identical species fuse together. Euploidy refers to the stance in which cells have a chromosomal number that is an integral multiple of the characteristic circum haploid number. Due to the common occurrence of polyploidy and aneuploidy in yeast, variable chromosome numbers elicit characteristics that may be beneficial in specific circumstances. As a result, the physiology and fitness of cells with different ploidy levels may differ. Bioinformatics is a highly interdisciplinary field that drives knowledge discovery from biological data using computational analysis. Today, bioinformatics is becoming an important part of most life science research. The process by which the DNA sequence of gene expression is copied into a gene product or RNA is explained by the central dogma of molecular biology. Microarray and more recently RNA sequencing; it has been widely used to measure gene expression levels. In this thesis, ploidy and genomic differences between the industrial Saccharomyces cerevisiae strain R625 and the freeze-thaw resistant evolved strain P8 obtained from R625 by evolutionary engineering were analyzed to gain insight into the complex molecular mechanisms of ploidy and freeze-thaw stress resistance.
Computational investigation of reaction mechanism of FET3 protein in yeast

(Graduate School, 2023-02-17) Ahıshalı, Büşra ; Balta, Bülent ; 521191104 ; Molecular Biology-Genetics and Biotechnology

In Saccharomyces cerevisiae, a yeast species, iron uptake into the cell takes place with the Reducing Iron Uptake Model. Ferric chelates (Fe3C-L) are degraded on the cell surface by being reduced from Fe3+ to Fe2+ by the cell surface reductases Fre1p and Fre2p. The free reduced Fe2+ ions are taken up by Fet3p-Ftr1p, a high-affinity oxidase-permease complex, or by Fet4, another metal carrier. In this study, the reaction mechanism and the role of Fet3p in reducing iron uptake are examined. Fet3p is a membrane-bound protein and a member of the multicopper oxidase protein family. It metabolizes iron uptake with a high affinity for Fe2+ and plays a role in iron uptake together with iron-permease Ftr1p. Since Ftr1p can only transport the oxidized form of iron, Fe2+ needs to be oxidized before entering the cell. Fet3p couples the four-electron reduction of O2 to H2O with the one-electron oxidation of four Fe2+. The oxidized iron leaves the iron-binding site in Fet3p and is transferred to Ftr1p. Thus, Fe3+ ions are transported into the cytoplasm by a permease, Ftr1p. The understanding of the mechanism of Fet3p is of great importance to shed light on other multicopper oxidase members such as laccases and human ceruloplasmin, some having wide industrial applications. When the active site structure of Fet3p is examined, it has 4 copper as a cofactor in the active site. These coppers are divided into 3 types according to their characteristics: Type 1 (T1), Type 2 (T2), and binuclear Type 3 (T3a and T3b). T2 and T3 coppers form the trinuclear cluster (TNC). Iron as a substrate is not observed in any of the crystal structures of Fet3p. However, according to the information obtained from mutation studies and comparing them with the crystal structures of other MCOs, especially copper efflux oxidase (CueO), the amino acids in the iron-binding region of Fet3p and the location of iron were determined. Fet3p couples four one-electron oxidations of 4 Fe2+ as a substrate to the four-electron reduction of dioxygen to water by taking four protons from the environment. This process is mediated by oxidation-reduction reactions of copper ions as cofactors and consists of two stages. In the first stage, the O2 molecule, which will be reduced to H2O during the reaction, enters the TNC through the solvent channel and binds to the TNC. The O-O bond is cleaved by taking two electrons from two coppers (T1 and one of T3 coppers). Proton donation of E481 to one of the oxygens bridging T3 coppers facilitates this cleavage. Finally, all coppers are oxidized to Cu2+, and one O2- and two OH- ions are formed. In the second stage, the four reductions from Cu2+ to Cu+ with oxidation of four Fe2+ to Fe3+, and four protonations occur, and OH- and O2- ions are converted to two water molecules. In the literature, most of the first stage of the reaction mechanism of MCOs, especially dioxygen-cleavage and peroxy intermediate structure are known. However, the exact mechanism of the second stage, the order of electron and proton transfer reactions is not known because this part occurs fast. Due to the rapidity of these reactions, they have not been studied before and the order of the reaction is unknown due to the difficulty of following the protonation order experimentally. In addition to examining the reaction scheme, it is known that D283 plays an important role in iron binding to substrate-binding site, and electron transfer (ET) is enhanced by D283. However, in the crystal structure, the loop containing D283 is oriented away from the active site, suggesting that it closes only after the binding of Fe2+. Thus, to find out the role of D283 on ET and reaction pathways, the geometries are separately examined when the loop containing D283 is open and closed. In order to elucidate the unknown parts, computational methods were used in this present study, so the possible reaction mechanism will be determined. Thus, it is aimed to understand the mechanisms of other multicopper oxidase members through Fet3p. The calculations and geometry optimizations were carried out using the Quantum Mechanics/Molecular Mechanics QM/MM approach. The M06-2X method, a Density Functional Theory (DFT) method, was used for quantum mechanical (QM) calculations. B3LYP, TPSS, and M06 methods were also used to investigate whether M06-2X is the most suitable method for energy calculations and geometry optimizations of Fet3p containing copper and iron metals. Although M06-2X is not recommended to be used on metals in the literature, all necessary electronic states and spin densities could be obtained only with M06-2X in this study. For this reason, the results were interpreted over the energies obtained with M06-2X. The determination of the QM region to be calculated during the QM/MM calculations is of great importance for the calculations to obtain more accurate results. While choosing the most ideal QM region, residues that have the potential to affect the reaction, electron transfer, and proton exchange, especially close to the region where the reaction took place, were determined. The proximity of amino acids that will contribute to electron transfer around copper and Fe was investigated; therefore, calculations were made accordingly by choosing different QM regions. Considering the computational costs, the most ideal QM region was determined. In the structure where the loop containing D283 is closed, the first Fe2+ oxidation occurs exothermically without protonation while T1 is reduced. Protonation of OH- or O2- ions are not needed due to the cost of protonation. It is examined whether the first electron transfers from T1 to TNC before the second iron binds; nevertheless, the structure could not be obtained without protonation. With the protonation of the TNC region, electron transfer to the TNC has yielded a stable structure. After the oxidized iron leaves, the second Fe2+ binds. Meanwhile, the electron already transferred from the first iron remains in the protonated TNC. Considering the necessity of a second proton transfer before oxidation, the proton taken from D94 returns back to D94 during the optimization, thus the second proton transfer is not necessary. The electron from Fe2+ transfers to T1 copper, and oxidation of the second iron takes place. The second oxidation, which was endothermic when D283 was open, is exothermic in the structure where the loop is closed. The results draw attention to the importance of the loop containing D283. After the second oxidation, the oxidized Fe3+ is replaced with the third Fe2+. For the third iron, structures with two protons, three protons, and four protons are examined. For the third and fourth Fe2+ the geometries when the loop with D283 is open are also examined. According to the results, even three protonations are not enough for third oxidation, and a fourth protonation is needed. When the loop containing D283 is open, the oxidation of the fourth Fe2+ is endothermic even in the presence of four protons in TNC, which is the maximum number of protons TNC can take. In the oxidation reactions protonation of TNC-O2- decrease the negativity of TNC; thus, electron transfer to TNC is more favorable. The protonation of TNC is important to reduce coppers at TNC (T2 and T3 coppers) and for transferring an electron from the substrate to TNC. Similarly, the transfer of an electron from the substrate to TNC and the reduction of TNC coppers force the TNC-O2- or T3-OH- to take proton.
Controlled release of tetracycline hydrochloride from silica based polycaprolactone nanohybrides

(Graduate School, 2022-12-23) Cengiz, Aybüke ; Güvenilir, Yüksel ; 521191130 ; Molecular Biology - Genetics and Biotechnology

Biomaterials can be synthetic or natural materials which is designed for interact with the biological systems. There are various type of biomaterials such as metals, seramics and polymers. Biodegradable biomaterials that naturally degrade or completely dissolve in their physiological environments have gained attention for both invasive and noninvasive health monitoring due to providing an unique opportunity for therapeutic field. Biodegradable polymers are classified into two main categories as natural and synthetic polymers. Polymers offer high adjustability in terms of their chemical structure and morphology and they contain hydrolysable bonds and these bonds makes them prone to chemical degradation via hydrolysis or enzymecatalyzed hydrolysis. Biodegradable polymers are using in controlled drug delivery, anticancer drug delivery, protein and peptide delivery, gene delivery, and enzyme immobilization at industry and researches. Biodegradable polymers can degrade in two main ways; partially or fully degrade to monomeric units. Polymers' degradation rate is heavily affected by various factors. Examples of these factors are the following such as the morphology, molecular weight, its distribution, crystallinity temperature and environmental conditions. Polylactic acid (PLA), polyglycolic acid (PGA), poly(lactic-co-glycolic acid (PLGA) and poly(ε-caprolactone) (PCL) are some of the most used synthetic biodegradable polymers. These are polyester polymers that have chemical structure with ester bond linkages and these polymers most used in industry are aliphatic polyesters. Polycaprolactone (PCL) is a linear aliphatic polyester hydrophobic semi-crystalline. PCL can synthesis with two different methods. These are the condensation of epsilon-hydroxycaproic acid and the ringopening polymerisation (ROP) of e-CL. In the condensation of epsilon-hydroxycaproic acid which is a polycondensation, 6-hydroxycaproic acid is polymerising by using lipase from Candida antarctica. ROP is the most preferred route and it gives a polymer with a higher molecular weight and a lower polydispersity. PCL is mainly synthesized by ring-opening polymerization (ROP) of ε-caprolactone (ε-CL) monomer. There are three different ROP catalytic system and metal-based catalysts are the most used catalysts for ROP of ε-CL. The metal based compounds takes part in the ROP of lactones can be describe as catalysts, initiators, initiating systems or catalytic systems. PCL have wide applicability and advantage such as biocompatibility, controlled degradability, miscibility with other polymers, and if its properties can be controlled and it can be made inexpensively, it can be a very useful polymer. Drug delivery systems (DDSs) are developed to prevent problems such as reducing therapeutic efficacy and causing unwanted side effects for improving drug safety and helping to improve patient compliance and convenience. DDS have many important applications in every field of medicine such as cancer, pain, diabetes and ischemia, myocardial treatment. Release pattern of DDS mainly effect by the delivery vehicle , the drug properties, and the environmental conditions. Drug delivery systems varies based on their route, mechanism and materials used in and there are several approaches of DDS. One of these approaches is polymers that used in a variety of fields in pharmaceutical applications. Polymers have advantages in DDSs as providing controlled release of therapeutic agents in constant doses over long periods and their cyclic dosage, and tunable release of both hydrophilic and hydrophobic drugs. In the first part of this study performed RHA preparation and activation of RHA, immobilization followed by polymerization to obtain nanohybrid polymers. Rice husk ash (RHA) is obtained by burning husks of rice. prepared and then silanized with 3-Glycidoxypropyltrimethoxysilane (3-GPTMS). RHA silanized by using 3-GPTMS as support material to immobilize free enzyme Candida antarctica lipase B. Enzymatic ring-opening polymerization (eROP) of ɛ-caprolactone was provided by immobilized lipase enzyme Candida antarctica lipase B (CALB). eROP reaction started along with ε-caprolactone and immobilized enzyme, then G-PCL/RHA nanohybrid obtained after terminating the reaction with chloroform followed by the precipitation. In the second part of the study microspheres prepared by trying different conditions to find best efficiency ones and followed by Drug release experiment to the highest effiency microspheres. Drug loaded microspheres were prepared by W/O/W double-emulsion-evaporation method which is water-in-oil method. Since the drug used in study is a water-soluble drug active substance, have to find the right ratio for both PVA and drug amount. Because of this, tried to find best PVA percentage by using different PVA percentages. Here found the best ratio is %1 PVA. Then looked at the drug amounts by preparing microspheres in %1 and different drug amounts. G-PCL/RHA drug-loaded microspheres with best drug loading efficiency we can get were obtained. Drug release experiments were carried out at pH 7.4 condition The release profiles of the drug-loaded G-PCL/RHA microspheres were determined and afterwards the drug release percentages are calculated. According to drug release results, the highest cumulative drug release percentage was %56 at pH 7.4 in %0.5 PVA, 10mg drug amount conditions microspheres with 72 hours burst level and %29 drug loading. Meanwhile the highest drug loading, efficiency, was in %1 PVA and 10mg drug amount conditions with %54 drug loading. In the last part of study, experiments has been finalized with charactization of microspheres to analyse microspheres by using Fourier transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA), differential scanning calorimetry (DSC) and scanning electron microscopy (SEM). FTIR spectra of G-PCL/RHA nanohybrid and drug-loaded G-PCL/RHA microspheres were compared along with literature tetracycline hydrochloride FT-IR spectra and the presence of tetracycline hydrochloride in the micropheres demonstrated. FT-IR results showed both G-PCL/RHA nanohybride and tetracycline FT-IR spectra on G-PCL/RHA microspheres FT-IR results. This proved the tetracycline hydrochloride presence on G-PCL/RHA nanohybrid microspheres. TGA results showed the decomposition temperatures and organic weight losses of drug-loaded G-PCL/RHA microspheres and tetracycline hydrochloride. According to DSC analysis, showed the melting and crystallization points of PCL polymer. Microsphere structures were observed by SEM analysis. Particles of the G-PCL/RHA microspheres were seen to be spherical and sphere-like structures with various size but they were more disadvantage cause of not being as highly porous compare to the literature microspheres. For better understanding drug-polymer interactions and higher how to get higher drug efficiency of microspheres on drug release mechanism, analysis methods can be developed in further studies. Beside drug release of hydrophylic drugs loaded microspheres can be carried out in vivo. Such studies exist in the literature.
DADA2 hastalarının periferik kan mononükleer hücrelerinde total ada aktivitesinin analizi

(Graduate School, 2022-02-09) Demirci, Turna ; Turanlı Tahir, Eda ; 521171122 ; Molecular Biology, Genetics, and Biotechnology

Deficiency of Adenosine Deaminase Type 2 is an autosomal recessive disease caused by biallelic mutations in the ADA2 gene. It was first defined as monogenic vasculitis syndrome in 2014 as a result of studies conducted by two different groups independently. Although it has been shown that the prevalence of ADA2 Deficiency maybe 4 in 100,000, the prevalence of the disease may differ between ethnic groups, depending on the degree of consanguinity and the presence of founding variants. Adenosine deaminase is an enzyme involved in the regulation of adenosine homeostasis and purine metabolism by converting adenosine to inosine and 2'-deoxyadenosine to 2'-deoxyinosine. There are two isoforms of adenosine deaminase in humans, and one of them, the 57-kDa homodimer ADA2 protein, is produced by the Adenosine Deaminase 2 (ADA2) gene. The N-terminal portion of the ADA2 protein is responsible for growth factor activity, while the C-terminal portion is responsible for adenosine deaminase activity. In addition to the catalytic domain, the ADA2 protein also has a protein dimerization domain and a cell surface binding domain. ADA2 proteins bind to different cell surfaces via glycosaminoglycan chains and to T cells via adenosine receptors. In this way, it shows both cytokine-like and autocrine-type growth factor properties. Although the ADA2 protein is involved in macrophage polarization, it also has an important regulatory function for neutrophil activation. In addition, it significantly reduces the formation of neutrophil extracellular traps, which are caused by extracellular adenosine and can lead to the activation of proinflammatory cytokines. Despite the clinical manifestations of DADA2 being very diverse, episodic clinical findings are usually observed in patients with fever and systemic inflammation. The most common type is vasculitis findings. In addition to dermatological and neurological symptoms, it is also rarely defined by renal involvement and gastrointestinal system findings. More than half of patients have attacks of non-infectious fever. Symptoms include recurrent oral and genital ulcers, musculoskeletal symptoms, recurrent abdominal pain, inflammatory bowel disease, and immunodeficiency. Hematologic findings include cytopenia, anemia, and rare bone marrow failure. The diagnosis of the disease is made based on the detection of pathogenic variants on the ADA2 gene or the measurement of ADA2 activity in serum/plasma. Treatment methods are selected depending on the symptoms and the severity of the disease. Currently, anti-TNF-α is the most common treatment modality, especially for patients with signs of vasculitis. Hematopoietic stem cell transplantation can be used in the treatment of hematological diseases. In addition, although it is not a suitable choice for long-term treatments, fresh frozen plasma infusions are also among the treatments applied. Enzyme-linked immunosorbent assay (ELISA) is a method used to detect and quantify protein in soluble substances, based on antigen-antibody interaction and measuring enzyme activity by colorimetric analysis. The purpose of this study was to compare the total adenosine deaminase (ADA) activity in peripheral blood mononuclear cells of patients diagnosed with DADA2 with the control group. 8 patients diagnosed with ADA2 deficiency and 5 healthy individuals were studied. Two of the patients are Syrian and have a G47R/G321E heterozygous mutation. 3 of the patients have G47R homozygous. Total ADA activity was measured in lysates prepared from subjects' peripheral blood mononuclear cells using a colorimetric ADA Activity Assay kit that is a commercial kit. ADA activity was calculated by following the protocol written in the kit, and then the statistical comparison of the results was analyzed by performing the t-test. The disease-causing variant p.G47R, which occurs in the dimerization domain, affects the stability of the homodimer required for enzyme activity of the ADA2 protein. Therefore, due to the decrease in ADA2 catalytic activity in patients with p.G47R mutation, it is expected that the total ADA activity will be lower than in the healthy group. As a result of statistical analysis, a significant difference was observed in ADA activity (p=0.0008). As expected, ADA activity was lower in the patient group compared to the healthy group. In addition, when patients with heterozygous mutations were compared with patients with homozygous mutations, lower ADA activity was observed in patients with heterozygous mutations. In this case, it can be said that the G321E mutation plays an important role in catalytic activity.
Detecting binding activity of a therapeutic monoclonal antibody targeting vascular endothelial growth factor using surface plasmon resonance

(Graduate School, 2024-07-25) Erdem, Serim ; Kılıç, Abdulhalim ; 521191137 ; Molecular Biology – Genetics and Biotechnology

The use of biopharmaceuticals, particularly monoclonal antibodies (mAbs), has become a major application following advancements in recombinant DNA technology. The treatment of a wide range of diseases, among them cancer, asthma, cardiovascular conditions, infections, inflammatory and autoimmune disorders, including allergies, has become possible with the development of mAbs. Surface plasmon resonance (SPR) is a well-known and reliable method that can evaluate biomolecular interactions in real-time in terms of binding affinity and kinetics without requiring labeling procedures. Using SPR technology to determine the kinetic parameters of a mAb molecule resulting from its binding with its target receptor is a common approach to characterize the binding activity of the final product. To characterize the binding kinetics between two molecules, one molecule is first stabilized on the sensor chip surface by immobilization or capturing steps. Then, the other molecule is flowed over the sensor chip surface at variety concentrations in a running buffer to observe the interaction between the two molecules and to determine the resulting kinetic parameters. In this study, after an IgG monoclonal antibody was captured on a Protein A chip surface, the target VEGF121 growth factor was flowed over the surface at five different concentrations. By employing single-cycle kinetic analysis, the kinetic parameters were measured, providing information about the interaction between the two molecules. To develop a robust kinetic assay, it was necessary to optimize several parameters, including the capture molecule (mAb) concentration, the concentration range of the VEGF121 molecule, dissociation time, regeneration time, and the established method sequence. While evaluating the robustness and reliability of the method, important factors considered included quality control parameters from the evaluation software, plotted residuals, and the consistency of Rmax and KD values between replicates. As a result of the method development studies, a capture concentration of 13.4 nM, a dissociation time of 240 seconds and a regeneration time of 30 seconds were decided upon, ensuring that the deviation in KD values between repeated studies was less than 20 %. After the KD value was obtained with low deviation between replicate studies, qualification studies including specificity, system precision, repeatability and intermediate precision parameters were initiated. As a result of 6 replicate studies conducted by two different analysts, the percentage difference between the analysts was determined to be 0.14%. Additionally, kinetic binding studies were performed with mAb molecules from 12 different production batches to demonstrate the effectiveness of the developed method in determining the binding activity of mAb molecules in different lots. These studies showed quite similar kinetic parameters for different production batches.
Evolutionary engineering of freeze-thaw stress-resistant yeasts without using chemical mutagenesis

(Graduate School, 2023-06-20) Balaban, İrem ; Çakar, Zeynep Petek ; 521211107 ; Molecular Biology - Genetics and Biotechnology

Saccharomyces cerevisiae, also known as budding yeast or baker's yeast is a unicellular microorganism from the fungi kingdom. It has been consistently used in winemaking, brewing and baking bread throughout human history. After 1930s, laboratory studies were conducted to obtain strains with increased product quality. Today, S. cerevisiae is the most popular yeast strain due to its good fermentative abilities. S. cerevisiae with high fermentation performance and tolerance to environmental stresses is preferred for industrial applications. During bread production, yeast cells are exposed to a variety of environmental stresses including freeze–thaw, high sugar concentrations, air-drying and oxidative stress. Stress conditions cause a decline in cell growth rate, product yield and quality. Cells give responses to stress conditions, as environmental stress response (ESR) and stress-specific response. ESR mechanism is not specific to the stress factor and it can be used to explain the cross-resistance of the yeast cells against various stress types. One of the reasons for cross-resistance is the use of the same transcription factors as a response to a variety of different stresses. S. cerevisiae is exposed to freeze-thaw stress during the cryopreservation and frozen dough process. Freeze-thaw stress causes physiological injuries to cells. At high freezing rates, formation of intracellular ice crystals causes cellular damages; while at low freezing rates formation of extracellular ice crystals causes cellular dehydration. The thawing process causes oxidative stress which leads to oxidative damage on proteins, nucleic acids and other biomolecules inside the cell. Studies conducted in S. cerevisiaes' stress-specific response against freeze-thaw stress revealed cells focus on regulating the contents of the cell membrane, protecting cell wall integrity, increasing degradation of damaged proteins from stress and increasing overall protein synthesis under stress conditions. Cryoprotective agents can be added to decrease ice crystal formation under freezing conditions. Alternatively, yeast levels in the product can be increased to increase product yield. However, these methods can decrease product quality and increase cost. Thus, stress-resistant S. cerevisiae strains are preferred for industrial applications. Stress-resistant strains can be obtained by metabolic engineering. Evolutionary engineering is an inverse metabolic engineering method that mimics the natural evolution process. In this approach, the desired phenotype is selected first and the genes responsible for the phenotype are determined later by reverse engineering methods. In this study, freeze-thaw resistant yeast strains were obtained with the evolutionary engineering method. A reference yeast strain was exposed to freeze-thaw stress in the form of pulse stress selection. The evolved strains obtained under stress conditions generally show mutations mainly in their stress-induced genes. This allows ease in reverse engineering studies to determine genes related to the applied stress. Freeze-thaw stress was applied in the form of pulse stress selection to maintain the survival rate of cells with increasing stress levels and to induce selective pressure. In this study, a S. cerevisiae CEN.PK113-7D reference strain was exposed to gradually increasing freeze-thaw stress until the final population was obtained. The final population was obtained after 10 cycles of freeze-thaw stress application. Ten mutant individuals were randomly selected from the final population and their resistance to freeze-thaw stress was tested with the spot assay method. Four evolved strains labeled as FT-1, FT-5, FT-6 and FT-9 that showed the highest freeze-thaw resistance were selected for detailed analysis. Further physiological characterizations of the evolved strains were made by cross resistance analysis. FT-1, FT-6 and FT-9 showed cross-resistance to potassium chloride (KCl) and iron stress. KCl, at high concentrations, causes hyperosmotic stress to the cell. This cross-resistance could be the result of a similar response mechanism activated by the cell to protect itself from dehydration caused by freezing stress. Metals such as iron increase generation of ROS in cell and cause oxidative stress. The cross resistance to iron stress could be the result of activation of similar pathways used by the cell as a response to oxidative stress caused by thawing process. All evolved strains tested showed resistance to boric acid. Boric acid disrupts cell wall synthesis in S. cerevisiae. The freezing process also causes cell wall damage in S. cerevisiae. Inducing cell wall synthesis due to freezing stress may also result with increased resistance to boric acid. The aim of this study was to obtain freeze-thaw stress-resistant S. cerevisiae strains from a reference laboratory strain, without using chemical mutagenesis, by evolutionary engineering. Physiological characterization of the evolved strains was also performed by determining their cross-resistance to selected stress factors. Further genomic, transcriptomic and proteomic analyses could be performed on the selected FT-9 strain to identify the genes, pathways and molecular mechanisms responsible for resistance against freeze-thaw stress and the pathways that cause cross-resistance to selected stress factors.
Evolutionary engineering of rapamycin-resistant yeast

(Graduate School, 2022-12-02) Esen, Ömer ; Çakar, Zeynep Petek ; 521191120 ; Molecular Biology-Genetics and Biotechnology

The budding yeast Saccharomyces cerevisiae, a unicellular eukaryotic microorganism, is widely used in many industrial processes such as baking, alcohol fermentation, biofuel and recombinant protein production as well as in basic research to understand the complex biological processes of advanced eukaryotic organisms, including humans due to its well-characterized genome and proteome, ease of growth and manipulation, as well as the similarity of its genes and pathways to higher organisms. Rapamycin is a macrolide compound that is produced by the bacterium Streptomyces hygroscopicus. In the medical field, rapamycin and its analogs are used to prevent organ transplant rejection, coat coronary stents and treat tumor cells. Many key metabolic pathways including cell growth and lifespan, protein synthesis and ribosome biogenesis, regulation of cell cycle and size, environmental stress response, nutrient uptake, starvation control, and autophagy in S. cerevisiae and also other higher eukaryotes are affected by rapamycin due to its inhibition of the target of rapamycin (TOR). Fkbp12 forms a complex with rapamycin that interacts with TOR resulting in its inhibition where the exact mechanism is still unknown. Fkbp12, immunophilin, is conserved in its structure and function among eukaryotes from yeasts to mammalians. Fpr1 found in S. cerevisiae is an orthologue of Fkbp12 found in humans. In this study, an inverse metabolic engineering strategy, evolutionary engineering was used to obtain rapamycin stress-resistant S. cerevisiae. Thus, serial batch cultivation of the S. cerevisiae CEN.PK113-7D reference strain under gradually increasing rapamycin stress was carried out. Before selection, a screening experiment was performed and 3 ng/ml rapamycin stress was determined as the initial stress level. During the selection process, the concentration of rapamycin in the medium was gradually increased from 3 ng/ml to 200 ng/ml over 61 daily passages or populations. From the final population, fifteen individual colonies were randomly selected which were named R1 to R15. Every individual (R1 to R15) was highly resistant to rapamycin stress in comparison to the reference strain (905). Afterward, R1, R3, R7, R12 and R14 were selected to continue for genetic stability test in which they were found to be genetically stable and their resistance to rapamycin was shown to be permanent. Genetically stable strains were tested by spot assay for their cross-resistance or sensitivity against various stress types, including 0.5 mM NiCl2, 2.5 mM CrCl3, 3 mM CoCl2, 17.5 mM MnCl2, 50 mM NH4Fe(SO4)2, 10 mM AlCl3, 20 mM CuCl2, 15 mM LiCl, 50 mM H3BO4, 100 µM AgNO3, 0.5 M NaCl, 15 mM caffeine, 4 mM vanillin, 200 ng/ml propolis, 1mM coniferyl aldehyde and 200 ng/ml cycloheximide. R12 was found to be the mutant with the highest number of cross-resistance and sensitivities: R12 strain was cross-resistant to CuCl2, NH4Fe(SO4)2, NaCl, coniferyl aldehyde, vanillin, cycloheximide, propolis; and sensitive to AlCl3, CoCl2, H3BO4 and AgNO3 in comparison to the reference strain (905). In order to determine the cell wall integrity of the rapamycin-resistant mutant (R12), lyticase susceptibility assay was performed. The result of this experiment showed that R12 resisted lyticase more than 905, under nonstress condition. On the other hand, under rapamycin stress, 905 resisted lyticase more than R12. Furthermore, the presence of rapamycin stress did not change the lyticase resistance of the evolved strain R12, in comparison to the nonstress condition. The chronological lifespan of rapamycin resistant strain (R12) was determined by using semi-quantitative and also quantitative chronological lifespan analysis. The result of the experiments correlated with each other in which R12 was found to have a shorter CLS, in comparison to 905. Comparative whole genome sequencing analysis of the rapamycin-resistant mutant (R12) revealed four single nucleotide variations (SNVs). These SNVs were located in four different genes. The precise functions of these genes in rapamycin response and resistance in yeast should be examined in greater detail in future studies. In conclusion, a rapamycin-stress-resistant and genetically stable S. cerevisiae strain (R12) was successfully obtained by using evolutionary engineering in this thesis study, and characterized at genomic and physiological levels. These results indicate that TOR pathway-related changes occurred in rapamycin-resistant mutant in order to overcome the high levels of rapamycin stress. However, in order to fully comprehend the molecular basis of rapamycin-resistance of R12, its comparative transcriptomic and metabolic analyses would be necessary as future studies.
Expression, purification and characterization of high-fidelity DNA polymerase

(Graduate School, 2022) Türk, Kübra ; Doğanay Dinler, Gizem ; 783309 ; Molecular Biology-Genetics and Biotechnology Programme

DNA polymerases found in all living cells discovered to date are the enzymes that synthesizes a new DNA strand complementary to template single stranded DNA. These enzymes do not only play a major role in the transmission of genetic information across generations during cell division, they also form the basis of Polymerase Chain Reaction (PCR), which is one of the most important in-vitro diagnostic techniques today. In addition to synthesis ability, DNA Polymerases may also have other properties including processivity, which is known as the ability of continuous polymerization, fidelity, which is known as the synthesis accuracy, and nucleotide selectivity. Thermostable DNA polymerase enzymes are mostly preferred in PCR-based studies because it is high importance that the stability of the enzymes used do not decrease depending on temperature. Taq DNA polymerase is the first discovered polymerase, which is a well-known enzyme used in a wide range of applications. Following the discovery of Taq DNA polymerase, the high-fidelity DNA polymerase was discovered in 1991 as a highly thermophilic DNA polymerase. Due to its high thermostability and proofreading properties, high-fidelity DNA polymerase is widely used in the applications that require high accuracy such as molecular cloning. High-fidelity DNA polymerase is an enzyme with a length of 775 amino acids and a molecular weight of about 90 kDa. This enzyme can perform 3'-5' exonuclease (proofreading) activity, which allows the addition of the correct nucleotides by removing the wrong nucleotides added to the structure during DNA synthesis. Due to this feature, it reduces the error rate during synthesis (1.3×10-6 mutations/base pairs/duplications), resulting in about 8 times less errors compared to Taq DNA polymerase. Many researchers have produced this protein by cloning it from Pyrococcus furiosus, a hyperthemophilic archaea, into different strains of Escherichia coli. The purification step is simplified by adding an affinity tag to the N- or C-terminus during the cloning. Based on these tags and various biophysical properties of the protein, purification protocols were created by affinity chromatography or ion exchange chromatography. In this study, we aimed to purify and characterize the high-fidelity DNA polymerase enzyme by taking the advantage of its thermal stability and 10X Polyhistidine-tag after bacterial production with high efficiency and low cost. For this purpose, commercially purchased pET16B. High-fidelity polymerase's plasmid DNA with a 10X Polyhistidine-tag at the N-terminus was used. The plasmid pET16B. High-fidelity polymerase was transformed into competent E. coli BL21(DE3) cells containing GroEL/GroES chaperonins to ensure soluble expression of the protein. In the first step of purification of High-fidelity DNA polymerase, which is a thermostable protein, all the folded proteins obtained from bacterial cells were heated and centrifuged to separate impurities with less thermal stability. High-fidelity DNA polymerase in soluble form was purified using IMAC affinity chromatography. The pure product was taken into a storage buffer containing 50% glycerol by filtration. The GroEL/GroES chaperonin system is a system that enables unfolded proteins with a molecular weight of 2-100 kDa to be folded in vitro and in vivo. Given that GroEL/GroES system can increase the folding of co-expressed recombinant proteins of different sizes by up to 70%, this system was employed in the production of High-fidelity DNA polymerase. However, while this system increases the amount of target protein, it can also increase the amount of impurities. Therefore, the purification of High-fidelity DNA polymerase was highly challenging. So that, various buffer compositions were used in order to optimize one step IMAC purification. Co-expression system was induced using IPTG for expression of pET16B. Expression of the polymerase regulated by the Lac operon. Since the growth temperature was chosen in the range of 12-20°C, where the metabolic rate of the cell and thus the growth rate was selected, the amount of protein folded by the chaperones was increased. Most of the impurities that increased with the target protein were eliminated with the 90°C heat treatment step. While heat sensitive proteins are eliminated from the environment, thermostable high-fidelity DNA polymerase enzyme, GroEL, and GroES proteins are still present. The separation of GroEL and GroES was achieved by applying IMAC affinity chromatography to increase the purity of the high-fidelity DNA polymerase enzyme. Purification results were analyzed by SDS-PAGE and immunoblotting methods. At the end of 500 ml bacterial production and purification process with three biological repetitions, high-fidelity DNA polymerase enzyme of similar quality with its commercial counterparts was produced, which can be used for a total of 60 000 PCR reactions with ~90% purity. The protein band on the SDS-PAGE gel was excised and analysed by peptide mapping using Liquid Chromatography-Mass Spectrometry (LC-MS) system to confirm that the produced protein is the target protein. According to the analysis, it was concluded that the purified protein was the target DNA polymerase. In order to determine if the purified protein is correctly folded or not, the secondary structure analysis of the protein with a purity over 90% was performed using Circular Dichroism (CD) in the far-UV (<260 nm) range. As a result of the study, the protein showed an apparent α-helix secondary structure with two minima at 208 and 222 nm wavelengths and a maximum at 190 nm wavelengths. Functional analysis of the protein on its folded state was completed by performing the Polymerase Chain Reaction (PCR). The 825 bp DNA region with 49.6% G-C content, and 1947 bp DNA region with 61% G-C content was amplified. These regions were selected considering the processivity of the enzyme. No-template control was used as negative control. The amplified regions were analyzed comparatively with commercial enzymes and it was observed that the target regions were successfully amplified. Commercially available high-fidelity DNA polymerase enzymes do not have endonuclease contamination and exonuclease contamination. Within the scope of quality control experiments, both endonuclease and exonuclease contamination of three biological replicates of our high-fidelity DNA polymerases were compared with commertial ones. λ DNA and λ DNA digested with HindIII were used as positive control. As a result, it has been shown that commercial enzymes and our high-fidelity DNA polymerase enzyme do not have endonuclease and exonuclease contamination. Another important test for demonstrating the quality of commercial enzymes is testing whether the protein remains stable under different conditions. For testing the stability of the polymerase, the effect of freeze-thaw stress repeated 20 times and also the effect of incubation of the enzyme for 5 days at room temperature were evaluated. As a result of these experiments, it has been shown that the freeze-thaw process during the general use of purified enzyme does not cause a negative effect on the activity of the protein, and that if the purified enzyme is forgotten at room temperature for up to 5 days during use, they can polymerize without reducing their activity. In conclusion, with this study, we have produced high-fidelity DNA polymerase with the same stability and processivity as commercial polymerases produced by large biotechnology companies with high efficiency and purity.
Expression, purification, and characterization of recombinant human IL-2

(Graduate School, 2022-01-18) Akgün, Buse ; Doğanay Dinler, Gizem ; 521181103 ; Molecular Biology – Genetics and Biotechnology

Cytokines, which are small proteins secreted by the immune system, are in charge of directing the immune system. Through their formation, differentiation, and activation functions, cytokines govern the maintenance of innate and adaptive immune responses. They are primarily formed by mononuclear phagocytes, dendritic cells, and antigen-presenting cells. Interleukin (IL) is a kind of cytokine that acts as an immunomodulatory protein. It induces a variety of cell and tissue responses. Interleukins mediate the interaction of leukocytes (white blood cells) and initiate a response by attaching to high-affinity receptors on the surface of the cells. They play a critical role in the regulation of cellular formation, differentiation, and activation that occurs over the course of inflammatory and immunological responses. Each family is assigned an IL based on sequence homology, receptor chain similarity, and functional qualities. Interleukin-2 (IL-2) was the first cytokine discovered to stimulate the growth of T lymphocytes. T cells, B cells, natural killer (NK) cells, lymphokine-activated killer cells, and macrophages all require IL-2 to regulate their proliferation and differentiation. Mier et al. discovered the molecule and named it "IL-2" since it was produced by and acted on leukocytes. Its discovery is regarded as a milestone in immunology. However, there is one issue that is common to all lymphokines when it comes to the molecular and functional characterization of IL-2, and it is due to their production in small quantities. The cloning of cDNA for IL-2 was a significant turning point in 1983, precipitated by the discovery of IL-2. The Jurkat T cell leukemia cell line was employed for the IL-2 cDNA clone development. IL-2 is a 15.5 kDa glycoprotein that belongs to the cytokine family four α-helical bundles. There are 153 amino acid residues in a single polypeptide chain of IL-2. IL-2 binds to and communicates with a receptor complex composed of three different subunits known as IL-2Rα (CD25), IL-2Rβ (CD122), and IL-2R (CD132). Different combinations of these three components bind to IL-2 with varying degrees of affinity. The αβγ heterotrimer, βγ dimer, and α chain monomer all bind to IL-2 with "high," "intermediate," and "low" affinity, respectively. Binding of IL-2 to the IL-2R heterodimer complex activates several pathways. In response to an interaction between interleukin-2 and its receptor, kinases connect to cytoplasmic areas of the receptor subunits, resulting in the tyrosine phosphorylation of many proteins and the activation of a number of signaling pathways, including JAK/STAT, PI-3K/AKT, and Ras/MAPK. IL-2 activity promotes cell survival, proliferation, cell cycle progression, and targeted gene transcription. Due to its ability to activate both T and NK cells, IL-2 was the first cytokine to be successfully used in cancer treatment. The US Food and Drug Administration authorized high-dose IL2 for the treatment of melanoma and renal cell carcinoma in xxii 1992 and 1998, respectively. Moreover, the use of recombinant IL-2 therapy may help researchers understand better the coronavirus disease 2019 (COVID-19), which is caused by a virus that leads to severe acute respiratory illnesses and has rapidly spread throughout the world. As a prospective treatment for this condition, the use of rIL2 may be beneficial for patients since it has the potential to accelerate disease recovery by increasing the number of lymphocytes in the body. A major difficulty is figuring out how to direct IL-2 activity toward Teffs and away from Tregs, which inhibit the immune system. IL-2 is available in two recombinant forms derived from E. coli, but only aldesleukin is FDA-approved. Recombinant IL-2 differs structurally from its natural version. IL-2 recombinant is not glycosylated and lacks N-terminal alanine. To avoid the formation of an incorrect disulfide bond, serine has been substituted with cysteine at amino acid position 125. The pharmacological actions of endogenous and recombinant human IL-2 are similar. In this study, E. coli Rosetta (DE3) was used as the host cell. Induction of protein expression was accomplished by the use of IPTG. Following that, inclusion bodies, which develop in the cell as a result of excessive protein expression, were separated and solubilized from cell lysates and refolded by step-wise dialysis. Anion exchange chromatography was used to separate the target protein from the rest of the protein mixture. After purification, the yield was determined to be 0.114 mg per liter of cell culture. SDS-PAGE and immunoblotting methods were used to validate the effectiveness of the purification. The molecular weight is estimated using intact mass analysis through LC/MS. The CE-SDS analysis revealed that rIL-2 has a purity of around 80%. In addition, the pI value of the protein was determined as 7.31 using the capillary isoelectric focusing method. The peptide mapping on LC-MS/MS is used to figure out the main structure of the protein that has been purified. The secondary structure of pure human interleukin-2 (hIL-2) was investigated using circular dichroism (CD), and the results revealed that it included a high concentration of alpha helices. The biological action of our IL-2 is determined by phosphorylation of one of the MAPK pathway proteins, extracellular signal-regulated kinase 1/2 (ERK), on human monocytic cells, THP-1. An active protein has been produced as a result of this work. The experimental results indicate that the procedures established for generating and purifying the rIL-2 protein may be employed to create a pure product that maintains its bioactivity.
Expression, purification, and characterization of soluble recombinant TNFR1

(Graduate School, 2022-06-28) Hatipoğlu, Derya ; Dinler Doğanay, Gizem ; 521191107 ; Molecular Biology – Genetics and Biotechnology

Tumor Necrosis Factor Alpha (TNF-α) is a trimeric cytokine secreted by macrophages and monocytes. It belongs to type II transmembrane protein family and is involved in both innate and adaptive immune system. TNF-α exists in two forms: transmembrane TNF (tmTNF), which is synthesized as a precursor form, and solubilized TNF (sTNF), which is created after further processing. TNF-α functions in a variety of biological processes by interacting with specific receptors namely as TNFR1 and TNFR2. TNFR1 consists of intracellular, transmembrane, and extracellular domains. The extracellular part consists of four cysteine-rich parts. When interacting to TNFR1, TNF-α can activate various signaling pathways for instance inflammation and apoptosis. TNF-α level is undetectable in normal individuals, yet under inflammation conditions the protein concentration in serum increases proportionally to the inflammation level in the body, thus making the protein detectable. Controlled production of TNF-α is crucial for tissue healing and fight against infection, but a constantly high level of TNF-α can cause various diseases including rheumatoid arthritis (RA), ankylosing spondylitis (AS), psoriasis/psoriatic arthritis, and Chron's disease. TNF-α inhibitors are designed and used in treatment of diseases caused by overexpression of TNF-α. An alternative pathway includes employment of TNFR1 extracellular domain as a biotherapeutic tool to inhibit the effects of overexpressed TNF-α. The latter approach seems to be way more preferred than other therapeutics due to its targetselectivity and high affinity. Production of proteins like TNFR1 are characterized by cysteine-rich domains by using bacterial systems is quite advantageous in terms of cost, yield, and time. However, lack of an effective translation system in Escherichia coli (E. coli) may hinder production of such disulfide bond containing proteins mainly in terms of generating mismatched cysteine residues or failing in acquiring a sulfide bridge formation thus leading to protein aggregation and inclusion body formation. Recovering proteins from inclusion bodies is time-consuming and expensive while considering the protein loss and incorrectly folded proteins due to the wrongly formed disulfide bonds. Dsb protein family including DsbA, DsbB, DsbC, DsbD, and DsbG, is responsible for the formation of disulfide bonds in bacteria. The DsbA-DsbB complex plays role in the oxidative pathways, while DsbC-DsbD complex functions in the isomerization pathway. Among all Dsb proteins, DsbA and DsbC are the most extensively studied and widely used in the field of biotechnology. Our previous studies showed that the extracellular portion of TNFR1 forms inclusion bodies in E. coli, and in vitro refolding steps are needed to obtain the correct xxvi conformation. Protein aggregations during the refolding process also caused a decrease in yield. The purpose of this study is to obtain the extracellular part of TNFR1 as a soluble protein in E. coli. Initially, a SHuffle T7 strain with a cytoplasmic DsbC copy was selected as the host cell and expression experiments were carried out accordingly. However, the target protein formed an inclusion body. To assess the effect of DsbC in the correctly folded-soluble TNFR1 production, a expression vector containing fusion protein DsbC-TNFR1 was constructed. Formation of DsbC-TNFR1 fusion protein was held on the basis of plasmid design conferring the order of a 6x histidine tag, DsbC, TEV cleavage site, and TNFR1 from N to C terminus respectively. It was assured that periplasm target sequence was removed from DsbC gene, allowing its production within the bacterial cytoplasm. DsbC-TNFR1 production was verified by SDS-PAGE and immunoblotting analyses, which revealed that some conditions led to the production of the fusion protein in soluble form. In order to detect the optimum conditions to produce DsbC-TNFR1, four different E. coli strains as host cells were employed, namely as Rosetta (DE3), Rosetta-gami 2, SHuffle T7, and BL21 (DE3). However, since production of the Rosetta (DE3) strain was higher than others, the research was conducted on that particular strain. Induction conditions including IPTG induction and autoinduction medium were also assessed to get higher yield from the selected cells. As a result of induction trials, the target protein was obtained in soluble form when autoinduction was utilized. Following sonication and centrifugation of the cells, the affinity chromatography was performed for separation of the recombinant protein from other host cell proteins. Anion exchange chromatography (AEX) was used as a second purification step to remove remaining impurities. The efficiency of purification was evaluated by using SDS-PAGE and immunoblotting analyses. Then, protein characterization studies were performed. The isoelectric point of the pure protein was calculated by isoelectric focusing via capillary electrophoresis device, and the purity of the protein was checked using the purity determination method. The secondary structure of the fusion protein was analyzed by circular dichroism spectroscopy and it was determined to be in the α-helix structure. Intact mass analysis through LC/MS was also performed to calculate the molecular weight of the protein. In addition, the peptide mapping was used to identify the amino acid sequence. The native structure of the pure protein was investigated by using blue native polyacrylamide gel electrophoresis (BN-PAGE), which revealed that the protein exists in both dimers and different oligomer structures. The functionality of the fusion protein was assessed by performing a pull-down assay, which showed that DsbC-TNFR1 is functional as it was capable of binding to TNFR1 ligand TNF-α. As a consequence of the research, it was revealed that the implementation of such a fusion protein is a successful tool for expressing TNFR1 in its soluble form in bacterial cells. After analysis of the pure protein generated as the outcome of downstream steps, it was shown that the TNFR1 produced by using the implemented fusion method is functional.
Functional analysis of vus (variant of uncertain significance) of human muts homolog 2/6 (hMSH2/6) proteins

(Graduate School, 2023-08-18) Gül, Celil Mert ; Doğanay Dinler, Gizem ; 521211103 ; Molecular Biology-Genetics & Biotechnology

Lynch syndrome (LS) or hereditary non-polyposis colorectal cancer (HNPCC) is a genetic condition that raises the risk of colorectal cancer and related cancers. Germline genetic variants of the DNA mismatch repair genes MSH2, MSH6, MLH1, and PMS2 are primarily responsible for its occurrence. In eukaryotes, MSH2 and MSH6 combine to form the MutSα complex, which is responsible for recognizing mismatches and assembling the necessary proteins for mismatch repair. Defining the functional consequences of variants is crucial in enrolling LS patients in appropriate surveillance programs to reduce morbidity and mortality. Herein, the mutation profile of hereditary colorectal cancer in the Turkish population was determined by analyzing the variation spectrum of 26 cancer susceptibility genes in 371 patients with colorectal cancer using next-generation sequencing technology. The detected variants were interpreted based on The American College of Medical Genetics and Genomics (ACMG) recommendations. The MSH2 and MSH6 loci were screened for variants of unknown significance (VUS) to determine the effect of nucleotide substitution on protein function. Mismatch repair deficient cell line (LoVo) was transiently transfected with hMSH2 wild-type (MSH2-WT) and hMSH6 wild type (MSH6-WT) genes and selected mutated subclones (MSH6-R577C, MSH6- S1279N, and MSH2-A733T). Regulation of mRNA expression and protein expression of interacting proteins were investigated, which showed hMSH6 gene expression was decreased when LoVo cells were transfected with MSH2-A733T compared to MSH2- WT. Expression levels of the downstream targets and interaction partner proteins were not affected significantly due to mutated forms of proteins. In-vitro binding assays showed that mutations did not affect the interaction between MSH2 and MSH6. Purified MSH2-WT and MSH2-A733T proteins that were produced from HEK293T cells were not obtained in stable forms. Bacterial co-expression of genes exhibited soluble protein production, and purification method for wild type proteins was promoted. However, due to the low stability of high molecular weighted proteins, it was decided that this method could be used in domain-specific studies with improvements. In order to reclassify clinical importance of the selected VUS, functional studies are needed to be improved. Reclassification of VUS in MSH2/MSH6 has the potential to improve variant classification accuracy, increase risk assessment, facilitate recommended clinical decision making, and provide more accurate genetic counseling for affected individuals and their families. Consequently, implementing personalized management strategies can effectively reduce cancer mortality and morbidity in Lynch syndrome populations.
Genomic analysis of freeze-thaw stress-resistant Saccharomyces cerevisiae

(Graduate School, 2024-07-12) Güney, Çağla ; Çakar, Zeynep Petek ; 521211104 ; Molecular Biology-Genetics and Biotechnology

The budding yeast Saccharomyces cerevisiae is a well-known unicellular eukaryotic organism widely used in industry for making bread and for ethanol production. In addition, it is highly used as a eukaryotic model organism for research purposes in molecular biology and genetics. Since its genome sequence is well-known for several years and it has homologous genes with other eukaryotes including humans, S. cerevisiae is used as model organism for studying higher eukaryotic organisms. Additional advantages of S. cerevisiae are its easy manipulation and well-known growth conditions. Freeze-thaw stress is a physical stress type, and freeze-thaw stress resistance is highly desired in S. cerevisiae. Freeze-thaw stress causes physiological damage to cell. Freezing step causes ice crystal formation and cellular dehydration and damages the cell. Thawing step creates damage as a result of ROS formation. S. cerevisiae is faced with this stress type while used in bakery industry. First, the dough prepared with yeast is frozen and stored. Then, the dough is thawed for the baking process. This would cause the drop of gassing power of the yeast. The drop in rising power is due to cyclic Adenosine Monophosphate (cAMP)-Protein Kinase A (PKA) pathway. There is a glucose-induced increase of cAMP which results in the activation of PKA. Then, trehalase breaks down the trehalose, which is an important component in yeast stress resistance, including resistant to freeze- thaw stress. Thus, freeze-thaw stress resistance is important for improving the efficiency of bread making process and other yeast bioprocesses. In this study, genomic and physiological analysis of previously obtained freeze-thaw stress-resistant S. cerevisiae strains were performed. The freeze-thaw stress-resistant mutant strains were obtained by an inverse metabolic engineering strategy, evolutionary engineering, without using any chemical mutagenesis. After batch selection, genetic stability of the mutants was determined to understand if the resistance to freeze-thaw stress was an adaptation, or it was caused by a permanent genomic change. The selected FT1, FT5, FT6 and FT9 mutant strains were found to be genetically stable. Determination of the cell wall integrity was done using lyticase susceptibility assay. The assay was performed with the genetically stable, freeze-thaw stress-resistant mutant FT9. Results of the lyticase susceptibility assay demonstrated that the freeze-thaw stress-resistant mutant FT9 was resistant to lyticase degradation more than the reference strain (905), under both stress and nonstress conditions. Comparative whole genome sequencing analysis of the freeze-thaw stress-resistant mutant (FT9) revealed only one single nucleotide variation (SNV). The SNV was located on CDC25 gene, and it was a missense SNV. As a result of the missense SNV on this gene encoding a cell division cycle (Cdc) protein, threonine was replaced by lysine (T1415K). In conclusion, a previously obtained freeze-thaw stress-resistant S. cerevisiae evolved strain was characterized in this study at genomic and physiological levels. The results revealed that cAMP/PKA pathway-related changes occurred in the freeze-thaw stress-resistant mutant strain in order to protect it against damage. However, in order to fully comprehend the molecular basis of freeze-thaw stress resistance of this strain, its comparative transcriptomic and metabolic analyses would be necessary as future studies.
Immunoreceptors modulate eosinophilic functions in viral immunity

(Graduate School, 2022-11-09) Durmuş, Lübeyne ; Muğan Çıracı, Ceren ; 521201116 ; Molecular Biology-Genetics and Biotechnology

Immunity a term used as resistance to pathogens, also referred to reactions of the body to noninfectious compounds such as tumors, harmless environmental substances and even sometimes host's components, each of which are called as tumor immunity, allergy and autoimmunity, respectively. The whole components like cells, tissues and molecules that generate this immunity is defined as the immune system and the immune response is coordinated by these components to foreign substances. Providing protection against infections or eradication of the infectious compounds from the body is the major physiological function of the immune system. Besides, growth of some tumors and cancers can be inhibited by stimulating immune reactions against cancer cells. The human defense system against pathogens can be divided into 3 levels: physical and chemical barriers, innate immunity and adaptive immunity. The innate immunity relies on a finite number of receptors for detecting the invading pathogens. These limited number of receptors target large groups of pathogens by recognizing conserved microbial patterns. Furthermore, activation of adaptive immune response is achieved by innate immune response. Innate immune cells include both hematopoietic and nonhematopoietic origin which make them different from adaptive immunity which relies on T and B lymphocytes. Innate immune response development involves hematopoietic cells that differentiate into monocytes, macrophages, mast cells, dendritic cells, natural killer cells, natural killer T cells, basophils, neutrophils and eosinophils. Moreover, hematopoietic cells include skin and epithelial cells reside in respiratory, genitourinary and gastrointestinal tracts. Innate immunity depends a few germline encodedreceptors which sense pathogen specific structural motifs. These receptors are collectively called pattern recognition receptors (PRRs) and microbial components recognized by PRRs are called pathogen associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). PRRs can exist in various cellular compartments like cell membrane and endosomal membranes, or cytosol. Moreover, they can be found in extracellular environment such as bloodstream or interstitial fluids. They are mainly divided into four major types: 1) Toll-like receptors (TLRs), 2) Nucleotide binding and oligomerization domain-like receptors (NLRs), 3) Retinoic acid-inducible gene 1-like receptors (RLRs), 4) C-type lectin receptors (CLRs). Activation of these receptors of the innate immune cells result with induction of adaptive immune cells. Hence, PRRs help to generate immune response to eradicate infectious agents, for example they induce the death of the infected cells. Humans are constantly under the threat ofinvasive opportunistic microorganisms including viruses, fungi, bacteria and parasites. The detection and development of an appropriate immune response against invading viruses are crucial for the outcome of virus infections. Recognizing viral nucleic acids is the first step for sensing virus infection. This recognition mainly depends on the genetically predetermined and germline encoded PRRs. There are TLR protein family members recognize viral genetic material such as TLR3, TLR7, TLR8 and TLR9. Upon recognition of viral nucleic acids or proteins by PRRs, production of type I IFN is induced resulting in the activation of target cells in both autocrine and paracrine manners. TLR7 and TLR8 have the ability to detect the existence of various single stranded RNA (ssRNA) viruses. These viruses include influenza, vesicular stomatitis virus (VSV), HIV, Sendai virus (SV) and a number of coronaviruses and flaviviruses. TLR3 is an endosomal nucleotide sensor which is located on endosomes and activated by double stranded RNA (dsRNA). As dsRNA is the genome for many viruses or they synthesize dsRNA during their life cycles, TLR3 also detects the existence of dsRNA and DNA viruses. Unlike TLRs, RLRs are RNA detectors locolized in the cytosol. RLR protein family includes three members: RIG-I, MDA5 and LGP2. Upon viral RNA association and oligomerization, RLRs bind to mitochondrial antiviral signaling protein (MAVS) through its CARD. MAVS has an essential role in RLR signal transduction as an adaptor protein whichinduces TBK1 and IKKε leading to the activation of IRF3 and IRF7. These transcription factors and NF-κB, together, activate the production of type 1 IFNs. Moreover, NLRs are important part of the cytosolic innate immunity and have a role in various key pathways such as inflammasome, MAPK, NF-κB and type 1 IFN signaling. Activation of inflammasome complexes is essential because it induces inflammation by leading to the production and secretion of IL1β and IL18 also known as inflammasome dependent cytokines. Eosinophils attract considerable attention with their identified roles in many pathological processes such as acute and chronic infections, cancer and thrombosis. Although their accepted roles in parasitic infections, involvement of eosinophils in fungal, bacterial and viral infections is an on-going research topic in the field of immunology. These granulocytes contain and generate substances with antiviral functions such as RNases and they may also involved in adaptive immunity with their potential antigen-presenting ability. Together, findings about eosinophils indicate potential antiviral role for eosinophils which need to be explored further. Despite the studies on animal models and primary human eosinophils demonstrating the importance of eosinophils against viral infections, the question of how eosinophilic functions are regulated following he viral infections is still ambiguous. Thus, the aim of the study is to investigate the changes in eosinophilic functions upon activation of TLR3, TLR7 and TLR8 with proper ligands, poly(I:C), R848, and ssRNA40, respectively. low number of eosinophils in blood (1-6%) makes them difficult to study in vitro. Therefore, we utilized EoL-1 human eosinophil line as a model in our study which was previously shown to serve an ideal model due to the expression of eosinophilic markers In this study we initially determined the activation of Eol-1 cells upon treatment with viral ligands, eosinophilic PRRs and immune receptors. Secondly, surface markers of eosinophils were determined to further understand the eosinophilic functions. Also, cytokines that they released were analyzed to understand the potential involvement in the induction of adaptive immunity. Moreover, production of matrix metalloproteinases was measured. We observed significant increase in IL-6 and IFN-γ secretion upon TLR8 activation. Also, the number of IL5Rα and PDL1 expressing Eol-1 cells were augmented with TLR3, TLR7/8 and TLR8 inductions. Our data suggested roles for TLR3, TLR7 and TLR8 in eosinophilic functions. We then investigated the changes in granule content of eosinophils during viral infections. We showed that ECP mRNA levels were upregulated upon the ssRNA40 treatment in Eol-1 cells. This data also, demonstrate the possible roles of ECP on the regulation of antiviral eosinophilic functions. MMPs are matrix-degrading enzymes which have roles in leukocyte recruitment to chemokines during microbial infections. In addition to their functions in immune regulation, MMPs can also lead to tissue damage as a result of persistant pathogen infections or spread. Furthermore, CD147 is a matrix metalloproteinase inducer that plays critical roles in various viral infections. Recently, CD147 was shown as an alternative receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Therefore, we also investigated the MMPs and MMP inducer CD147to better understand their roles in eosinophilic antiviral immune responses. The results from RT-qPCR showed a relationship between both CD147 and TLR activation as well as the data obtained from gelatin zymography clearly indicated the relationship between MMP-9 and TLR activation in Eol-1 cells. All stimulants tested in this study elevatedthe CD147 at mRNA level in Eol-1 cells. Additionally, TLR3 and TLR8 ligands significantly increased the Pro-MMP9 production at 24 hours post-induction in EoL-1 cells. Upregulation of TLR8 led us investigate the potential adaptor molecules which may have roles in downstream signaling pathways through TLRs in eosinophils. CARD9 is a multifunctional adaptor protein which participates in different phases of the immune system including fungal, bacterial and viral infections. Thus, we investigated the CARD9 expression in Eol-1 cells upon TLR3, TLR7/8 and TLR8 inductions. Despite the decreased mRNA expressions of CARD9, there was a significant increase in CARD9 expression at protein level in Eol-1 cells. Next, we measured the expression levels of cytosolic PRR RIG-I at protein level because of its well-known roles in antiviral immunity. We observed 1.49-fold increase in the cells stimulated with TLR8 agonist (ssRNA40, 2 μg/ml). Then, we measured NOD2 as it is already known as the interaction partner for CARD9. Numerous studies formerly showed the NOD2 involvement in response to many RNA virus infections such as parainfluenza virus, VSV, RSV and IAV. Here we showed that NOD2 protein expression was significantly increased after induction with TLR7/8 and TLR8 ligands in Eol-1 cells. Overall, of all the stimuli we tested, ssRNA40 (TLR8 signaling) was the most potent in inducing the mRNA expressions of ECP and CD147, and protein expression of immune receptors, surface markers of eosinophils and cytokines. Interestingly, CARD9 which is a critical adaptor against fungi infections was significantly increased after induction with TLR7/8 ligands, suggesting an important role for TLR7 and TLR8 rather than TLR3 in antiviral immune response generated by eosinophils. Our findings will pave the way for future studies focusing on eosinophil related infectious diseases.
Improving bone tissue integration of hard tissue implamants using bioactive materials

(Graduate School, 2022) Kerem, Gizem ; Kılıç, Abdulhalim ; 867049 ; Department of Molecular Biology-Genetics and Biotechnology

Hard tissue implants which made of different materials are used widely in medical applications in bone and tooth deficiencies. Metallic and ceramic materials are preferred as hard tissue implants because of their strength and toughness. Due to low density, high corrosion resistance and biocompatibility properties of titanium and titanium alloys, their usage as an implant has gradually increased. Implant materials should not cause an immunogenic reaction in the body and also in recent years thanks to new technologies in biomaterial field there are some applications to improve interaction in a positive way between implant and implantation tissue. Some morphological changing applications like grooving and acid etching, some physicochemical activation treatments like hydroxyapatite (HA) coating and titanium oxide (TiO2) coating, some biochemical activation treatments like coating with biopolymers and immobilization of bone morphogenic proteins and inducer chemicals are studied on titanium surface to improve bone formation, bone bonding and antibacterial property. At this study, titanium (grade 2) surfaces are chosen to increase its bone tissue integration by designing bioactive surface by using chitosan microspheres as a natural polymer and water-soluble dexamethasone (DEX) as an inducer for SAOS-2 cell differentiation. Chitosan microspheres were produced by using single emulsion-crosslinking method. Different amount of glutaraldehyde with different percentages was used as a crosslinker agent to obtain optimum DEX loading into chitosan microspheres. Crosslinking time parameter was also changed for optimization. Dexamethasone loading was tried in two ways first one is adding drug into dissolved chitosan solution then produce chitosan microspheres, second one is adding produced chitosan microspheres into the drug solution. DEX was loaded into chitosan microspheres via diffusion. After optimization process, chitosan microspheres were produced by using glutaraldehyde as a crosslinker. Chitosan microspheres were placed into DEX solution for loading process. Drug release studies were performed by using DEX-loaded chitosan microspheres, and released amount of DEX was determined. Drug loading efficiency was found as 50.16% and release of DEX was observed for 12 hours and the released amount of the loaded DEX was calculated as ~32.6%. Before chitosan microsphere coating onto titanium surfaces, they were treated chemically (polished, oxidized and silanized to produce amino groups on titanium surfaces). Then the samples were activated by glutaraldehyde and coated with chitosan microspheres.
In vitro and in silico investigation of NFIB-SUMO interactions

(Graduate School, 2022) Özkan, Ayberk ; Kumbasar, Aslı ; 718174 ; Molecular Biology-Genetics and Biotechnology Programme

Nuclear Factor I family of transcription factors are involved in regulation in diverse physiological processes, including neuronal terminal differentiation, gliogenesis, stem cell quiescence as well as in pathologies such as tumorigenesis and cancer progression. NFI family is encoded by four genes: NFIA, NFIB, NFIC and NFIX. NFI proteins contain a highly conserved N-terminal DNA binding and dimerization domain, while their C-terminal domains diverge. Because of their highly similar DNA binding and dimerization domain, NFIs bind to a palindromic consensus site with similar affinity in vitro, potentially regulating the same set of target genes in vivo. Any functional differences between the family members may arise from the more diverse C-terminal domain provides which can promote either transcriptional activation or repression. NFIs may regulate target gene expression via different mechanisms of action. NFI can bind directly to DNA and regulate the expression of the target gene or interact with another protein to affect gene expression indirectly. Moreover, NFIs can interact with histone proteins and cause alterations in the nucleosome structure, thereby being involved in the formation of the transcription complex. NFIs can directly interact with and facilitate recruitment of basal transcription factors. NFIs can also bind co-activator or co-repressors to control transcriptional activation. In addition, NFIs can, along with other transcription factors, co-regulate target gene expression. Finally, NFIs can promote dissociation of DNA methyltransferase from target gene promoters and activate transcription. Any alterations in the production or action mechanisms of NFI proteins lead to important developmental defects as well as cancer. One member of the NFI family, NFIB, is an essential gene as demonstrated by studies on knockout on mice: silencing of NFIB leads to perinatal death due to lung defects. NFIB controls stem cell differentiation in different cell types such as, adipocytes, megakaryocytes, melanocytes and hippocampal neural progenitors. Interestingly, in humans, mutation of one copy of the NFIB gene can result in intellectual disability and brain malformations. These findings underscore the importance of NFIB as a transcriptional regulator, however, the mechanism by which NFIB acts or regulatory events upstream of NFIB have not been fully elucidated. Indeed, scarce data exists regarding NFI post-translational modifications. Phosphorylation, glycosylation, acetylation, sumoylation may regulate the activity of NFI. Among these modifications, sumoylation is conserved by eukaryotic organisms. Sumoylation regulates many cellular mechanisms such as nuclear transport, chromosome segregation, and transcription activation/repression. SUMO (small ubiquitin like modifiers), which is generally observed as a suppressor in transcriptional regulation, can be conjugated to many transcription factors and affects the activity of these factors. The SUMO gene family has five mammalian isoforms: SUMO1, SUMO2, SUMO3, SUMO4 and SUMO5. SUMO peptides are activated by a series of enzymatic processes. These processes are required to form mature SUMO, which is active and may able to conjugate specifically to the target protein. The sumoylation consensus sites and SIM (SUMO interacting motif) on target proteins enable SUMO to specifically recognize and bind to these proteins and regulate their activities. Sumoylation can affect transcription factors in several ways. SUMO can compete with other modifications, may interact with co-activators, and can control the binding of the transcription factor to its target site on chromatin. In addition, sumoylation can control intracellular localization of transcription factors. Sumoylation of NFI has been shown in vitro. Interestingly, a study on neuroblastoma cells exposed to oxidative stress, identified NFIB among sumoylated proteins modified on sumoylation consensus sites. However, sumoylation of NFIs have not been further explored, in silico and in cell culture. In this study, we set out to investigate the functionality of sumoylation consensus site and SUMO interacting motifs of NFIB, using in silico methods and forced expression in cell culture. Currently, there is no experimental or modeled 3D structure of NFI proteins. Information about NFI protein structure is quite limited. As mentioned above, NFI proteins contain an N-terminal DNA binding and dimerization domain and C-terminal transactivation domain. NFI proteins carry four conserved cysteine residues in their DNA binding and dimerization domains, three of which are required for DNA binding activity. Another piece of evidence regarding NFI structure comes from the homology with the MH1 domain of SMAD3. Both proteins have highly similar Cys-His box motifs consisting of three cysteine residues and one histidine residue. Nevertheless, this homology is below 30%. Here, due to the lack of high homology and also fold similarity, we used ab initio modeling method to predict structure of NFIB DNA binding and dimerization domain. Subsequently, these predictions were compared to each other. For this comparison, we focused on the cysteine residues which are required for DNA binding activity as well as the conserved MH1 Cys-His box motif. Then, selected structure prediction models were assessed by molecular dynamic simulations. Finally, with REMD (Replica Exchange Molecular Dynamics) the model that showed higher stability and quality was verified. We performed molecular docking simulations to investigate NFIB SIM-SUMO1 interactions. We found that SUMO1 would preferentially bind to a specific NFIB SIM. Meanwhile, to investigate NFIB-SUMO1 conjugation in vitro; site-directed mutagenesis was performed for generation of a sumoylation consensus site mutant and a SIM mutant. HEK293T cells were co-transfected with SUMO1 and wild-type NFIB or NFIB mutants and NFIB was immunoprecipitated for analysis of NFIB-SUMO1 conjugation. Future experiments are required to validate the putative NFIB sumoylation consensus site and SIMs in cell culture.

Gözat

Başlık ile LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji-Yüksek Lisans'a göz atma

Sayfa başına sonuç

Sıralama Seçenekleri