LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji-Yüksek Lisans

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  • Öge
    The synthesis, SLIC based labeling, and characterization of microbial rhodopsins by using custom build spectroscopic methods
    (Graduate School, 2023-01-26) Çavdar, Cansu ; Bayraktar, Halil ; 521191105 ; Molecular Biology-Genetics and Biotechnology
    Type I opsins (also known as microbial opsins) are seven transmembrane-domain proteins with retinal chromophore absorbing incoming light. Most of them are ion channels or pumps although they do not directly bind to G protein complexes. They are found in all three domains of life. Numerous homologous forms of rhodopsins have been identified in the microorganisms, including light sensors (sensory rhodopsins), transmembrane chloride pumps (halorhodopsins), and energy saving transmembrane proton pumps (bacteriorhodopsin or proteorhodopsin). Rhodopsin proteins are widely used in the field of biotechnology. For example, it is used to determine the membrane voltage level in neurons. The use of rhodopsins as tools to control membrane potential with light is another technique for transformative optogenetics technology. Membrane voltage is present in all cells, and it creates an electric signal to carry the signal across the cell membrane and provides cell-cell communication. Since the absorption methods are not sufficient to measure voltage signal due to low signal to noise ratio in cells, more sensitive fluorescent methods based on rhodopsin are strongly preferred for a wide spectrum of applications. An understanding of the dynamics of the microbial rhodopsin proteins is essential to tune the photophysical properties of rhodopsins. It is also necessary to label them with fluorescent proteins and characterize their localization in detail. For the fluorescent signal to vary with the amount of light absorption in the membrane protein, the linker peptide between the proteins has to be optimized for various applications. For electrochromic fluorescent energy transfer, it is necessary to select the appropriate fluorescent protein. The emission signal of the fluorescent protein must overlap with the absorption signal of the membrane protein. After the most suitable fluorescent proteins are selected by calculating the amount of overlap, the structure of the peptide that binds the membrane and the fluorescent protein should be determined. Since both the length and the bending ratio of the selected peptide are important, it is necessary to optimize by testing different constructs. Here we have synthesized, purified, and studied the rhodopsin by using molecular biology and various spectroscopy methods. After the synthesis of rhodospins in BL21 cells, it was purified with his-tag affinity column chromatography and reconstituted with a detergent solution. The color tuning of rhodopsin as a function of pH was investigated by using absorption spectroscopy. We found that BPR undergoes a large red shift under acidic conditions. A pH value was increased the color turned from orange to red at the basic solution. We concluded that the deprotonation of the retinal at the rhodopsin center results in a significant change in the color of BPR. We have also measured the transient absorption changes of BPR by using a custom home built spectrometer that was equipped with two laser lines and an op amp light detector. The data acquisition and the control of lasers were performed an by arduino and field programmable gate array device programmed with arduino and labview respectively. Our results indicate that BPR underwent an absorption change after stimulated with a 532 nm diode laser. Finally, the fluorescent proteins were also cloned into the SRII gene by using SLIC cloning method and expressed in BL21 cells to determine the changes in fluorescent emission. Sensory rhodopsin was similarly characterized by using absorption spectroscopy. As conclusion, BPR undergoes a large spectral shift due to deprotonation upon decreasing pH and alters the color of the protein. SLIC method provides a cost-efficient method to prepare fluorescently labeled rhodopsin proteins. Contrary to the standard cloning techniques used in molecular biology, the SLIC method, which is convenient in terms of time and cost, has been studied and the method has been optimized. The optimized SLIC method can be used as an alternative to other molecular cloning techniques. The custom build pump-probe system can also be used for the characterization of fluorescently labeled other rhodopsin proteins in future studies.
  • Öge
    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.
  • Öge
    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.
  • Öge
    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.
  • Öge
    Inverse metabolic engineering of KCl-resistant Saccharomyces cerevisiae
    (Graduate School, 2022-06-20) Morkoç, Ogün ; Çakar, Zeynep Petek ; 521181116 ; Molecular Biology - Genetics and Biotechnology
    Bu çalışmada, KCl tuzu kaynaklı hiperosmotik strese dayanıklı S. cerevisiae suşları elde etmek için, tersine metabolik mühendislik yaklaşımı olan evrimsel mühendislik kullanılmıştır. Evrimsel mühendislik ile elde edilen suşların fizyolojik ve metabolik analizleri yapılmıştır. Evrimsel mühendislik için seçilim deneyi, kademeli olarak artan KCl stresi altında gerçekleştirilmiştir.