FBE- Moleküler Biyoloji-Genetik ve Biyoteknoloji Lisansüstü Programı - Doktora
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ÖgeDevelopment and structural determination of antiangiogenic recombinant antibody structures for cancer treatment(Lisansüstü Eğitim Enstitüsü, 2022)Vascular endothelial growth factor (VEGF) is a signaling molecule that plays a role in both vasculogenesis and angiogenesis. VEGFR-2/KDR (vascular endothelial growth factor receptor 2/ kinase insert domain receptor) is a tyrosine kinase that regulates a variety of angiogenesis-related cell responses. VEGFR-2/KDR is located on the surface of tumor cells and is heavily expressed in tumor-associated endothelial cells, where it modulates tumor-promoting angiogenesis. The suppression of VEGFR-2, which inhibits the angiogenic pathway in carcinogenesis, is thought to be a viable therapeutic method for the prevention and control of solid tumor growth. There are currently antibody (Ab) therapeutics targeting VEGFR-2 to treat cancer cells, but none of them is a final cure. Expectedly, the production of a novel effective anti-VEGFR-2 Abs would support cancer patients. The advancement of recombinant antibody technology and their tailored fragments has resulted in the emergence of a diverse array of antibody molecules, including the antigen binding fragment (Fab), the variable fragment (Fv), and the single chain variable fragment (scFv). Compared to the large size of a full antibody, scFvs have greater tissue penetration, decreased immunogenicity due to small molecular masses and accelerated clearance time. However, there are also some drawbacks of production of scFv structure which has poor biophysical properties, such as increased aggregation tendency and decreased thermodynamic stability, resulting in low performance and application. Protein solubility is critical in the synthesis and administration of therapeutic proteins. Inadequate solubility can lead protein aggregation, a detrimental phenomenon that can occur at any stage of recombinant protein synthesis. Numerous methods, extending from culture conditions to solubility enhancer tags, are being used to increase soluble expression. While fusing proteins to mostly soluble companions or expression with molecular chaperones may increase their total solubility, these techniques typically impair the proteins biological activity. There is no universal solution or technique for protein aggregation. anti-KDR 1.3 and anti-KDR 2.6 (anti-VEGFR-2) scFv molecules were formerly developed by phage display technique by TUBITAK MAM GMBE Immunogenetics Laboratory group. However, these antibodies are produced in inclusion bodies and form protein aggregates. Additionally, folding these molecules into active biological molecules is a laborious and uncertain process. Additionally, the protein yields are usually poor. The aim of this thesis is to address the expression and solubility problem of anti-KDR scFV molecules and to confirm the anti-angiogenic activities of the produced target molecules. In this manner, two different approaches have been adopted in the thesis, the first is the production and control of the target scFv molecules in mammalian cells, and the second approach is to transfer the target scFv molecules to a different structure using the complementarity determining regions CDR-grafting method, ensuring their production in bacteria and controlling their activity. In the first approach, the target scFv protein expression in mammalian cell culture was investigated. The existing scFv genes were utilized in the research. Different signal peptides were used for the construction of the plasmids including the target genes. Simultaneously, codon optimizations for the target cell line based on the target genes were performed. For this study, newly developed scFv genes were cloned into different mammalian expression vectors and transferred into competent cells by chemical transformation. Then, the cloned vectors were determined by colony polymerase chain reaction ( PCR) studies. The DNA sequences of the newly cloned scFv constructs were controlled by a capillary electrophoresis system. With the results obtained, sequence analysis was performed, and the proper incorporation of the gene into the plasmid, as well as the presence of mutations, were examined. The sequence of the target genest obtained were translated using bioinformatics tools, and the protein to be generated was examined for frameshifts. In accordance with these findings, the resulting vectors were purified and transfected into mammalian cells. New scFv constructs were produced in mammalian cells and purification was made by using chromatography columns. Transient transfections with vectors encoding the regulated target genes were initially conducted in different mammalian cell lines. Then, stable transfections were generated using the optimum antibiotic concentrations. The serial dilution technique was used to produce monoclonal cells from stably transfected cells. Chinese Hamster Ovary (CHO-K1) cells were adapted and suspended in serum-free media. Several feeding strategies, temperature experiments, and high volume production experimets were carried out in the cell culture studies. The resulting antibody structures were analyzed by SDS-PAGE and western blot methods. The binding of the samples to VEGFR-2 were then be checked by ELISA assays. After production and purification of the proteins, the activity and structure analysis were performed. Purified antibodies were examined by chromatographic methods based on HPLC (High Performance Liquid Chromatography). Real-time PCR method was used to investigate the presence of m-RNA in cells. In addition to these studies, Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS) studies were carried out to reveal the structural dynamics of the anti-KDR 1.3 protein obtained from bacteria. Unfortunately, it was not possible to achieve better expression efficiency for the target anti-KDR molecules in mammalian cells. Overall results for poor expression or none were confirmed by monoclonal cells experiments, fed-batch experiments, codon optimizations, signal peptide alternatives. The selection of high-product yielding clones was requiring significant amount of time and effort in the development of cells producing recombinant molecules. As a result, the second half of this thesis focused on developing a unique technique for the quick and simple increase in solubility and production of the target proteins. In the second approach, a CDR grafting technique was used to modify and upgrade the structure of the existing scFv with a more producible pre-existing scFv to improve the biophysical characteristics of these scFv molecules. The effect of the CDR grafting technique on the solubility and efficacy of the expressed protein was investigated in this study. Computational methods were used to model our antibody structure and physicochemical properties, followed by experimental confirmation. CDR grafting technique was employed to modify and upgrade the structure of our scFv with a more producible pre-existing scFv. We used computational methods to model our antibody structure and its physicochemical properties, followed by experimental confirmation. Soluble expression analysis, affinity determination by surface plasmon resonance (SPR), in vitro activity assays were performed. According to the computational data, CDR grafting enhanced the solubility of the grafted scFv protein in comparison to the native scFv protein. SDS-PAGE and western blot analysis showed the expressed protein's increased solubility. SPR analysis revealed that the grafted molecule exhibits comparable binding affinities to the VEGFR-2 receptor to the native molecule. Biological activity studies, including proliferation inhibition, migration and wound healing experiments studies on human umbilical vein endothelial cells (HUVEC), demonstrated that the newly synthesized grafted scFv protein posseses anti-angiogenic characteristics. This study reveals that a soluble scFv can be created by grafting the variable regions from an intrinsically insoluble scFv onto constant sections derived from innately soluble molecule. The drug potency of a low yield anti-angiogenic scFv (anti-KDR 1.3) with inclusion body formation tendency was highly increased by employing CDR grafting strategy.
ÖgeMolecular characterization of silver-resistant Saccharomyces cerevisiae(Lisansüstü Eğitim Enstitüsü, 2021)In molecular biology and metabolic engineering, one of the most widely used organisms is the yeast Saccharomyces cerevisiae that has been involved in biological applications for thousands of years among different cultures in different parts of the world. As a eukaryotic organism, S. cerevisiae reproduces by budding or sporulation, as a consequence of being able to have genetically haploid and diploid forms. It has a high level of similarity with human cells in regard of proteins and their functions, which leads to the popularity of this budding yeast in a wide range of researches related to humans. Yeast can resist many types of stress factors in the environment at various levels. Metal stress is one of these stresses that yeast may show resistance against, but the mechanism of metal stress tolerance in yeast has not been fully enlightened yet. When these metal resistance pathways are revealed, it will be also possible to increase resistance, which leads to an improved productivity in metal recovery in industrial applications for biotechnological processes and in bioremediation of metal-polluted environments. Silver stress resistance in yeast was studied in this thesis and after obtaining a silver-resistant S. cerevisiae mutant by using evolutionary engineering methods, the mutant was subjected to genetic and physiological characterization by comparison with the reference strain. Ethyl methanesulfonate (EMS) treated S. cerevisiae was the initial culture in this study, and this chemical mutagenesis caused a wide range of random mutations in the genome of the yeast cells in the population. To select a target mutant with the character of silver-resistance in the population, evolutionary engineering method was applied. To determine the initial level of silver stress for the evolutionary selection, this culture and the reference strain were both cultivated at different silver concentrations. The survival rates calculated at different silver concentrations demonstrated the most appropriate initial silver concentration with the minimum effect of silver stress on the cells. Next populations were subjected to higher concentrations of silver than the previous ones, until a significant decrease in the survival rate. While 5 µM AgNO3 was the initial stress level for the first population of the selection, the 29th population was exposed to 250 µM AgNO3. Ten individual mutants were randomly chosen from the final population, based on their high resistance to silver stress. Among these mutants, the most resistant one, called as 2E, was tested for genetic stability that revealed a genetically stable trait. The silver-resistant mutant 2E and the reference strain were subjected to cross-resistance tests against other stresses, using spot assay. Spot assay results showed that 2E was also highly resistant to copper stress and had a significant resistance against cobalt and oxidative (H2O2) stresses. On the other hand, it was observed that 2E was sensitive against chromium, manganese, aluminum, and ethanol stress. Reference strain and the silver-resistant mutant 2E showed similar stress responses against nickel, zinc, and lithium stresses. Transcriptomic analysis for the whole genome of the silver-resistant mutant 2E and the reference strain was applied to investigate the mechanism of silver resistance in the cell. DNA microarray analysis revealed that while 780 genes were upregulated, 877 genes were downregulated. Totally 1657 open reading frames (ORFs) had differential expression between 2E and the reference strain. By using Gene Ontology analysis, it was indicated that in the silver-resistant mutant, the highly upregulated genes were related to carbohydrate metabolic process, precursor metabolite generation, energy generation, oxidation-reduction, oxidative stress, and extracellular stimulus response. On the other hand, mutant 2E seemed to have generally repressed its protein synthesis by downregulation of the genes involved in ribosome biogenesis, ribosomal subunit and RNA metabolic processes. When compared to the reference strain, there were 64 mutations in the silver resistant mutant 2E, based on the results of whole genome re-sequencing. These mutations were composed of 61 nonsynonymous and 3 stop-loss ones. The most relevant mutation in 2E that may have caused an improvement in silver resistance was the one on CCC1 gene, a transporter of Fe2+/Mn2+ in vacuole, since silver has similar atomic properties with copper. This study also implied that the resistance of 2E against silver might be by upregulating copper resistance genes, CTR3, CUP2, CUP1-1, and CUP1-2, that bind copper and are responsible for copper-homeostasis in the yeast cell. Silver causes oxidative stress in cell and the mutant 2E had oxidative stress resistance. This study showed that a number of oxidative stress responsive genes were oppositely expressed in the mutant 2E and the reference strain that showed no resistance against neither silver nor oxidative stress. Oxidative stress resistance of 2E was also supported by the missense mutation detected on the DNA helicase gene PIF1. In addition to this, 2E had a highly differentiated mitochondrial gene expression profile that led to a more active aerobic metabolism and electron transfer system, which may explain the better growth of the mutant 2E under silver stress condition. The membrane-bound transport and cell wall proteins are damaged when subjected to silver ions and nanoparticles during growth phase, that leads to disruption in cell wall and membrane integrity. However, while 2E upregulated some of the mannoprotein genes (such as YPK2, USV1, YPS6, SRL1) to keep its cell wall integrity, it downregulated the ones related to anaerobic growth (TIP1 and TIR1-4 genes). Additionally, some ergosterol synthesis genes and many of the sugar transporter genes along with some TCA cycle genes for NADH regeneration were significantly upregulated. All these findings indicated a strong aerobic metabolism in the evolved strain 2E, but a more robust cell wall was most likely provided by the upregulation of other cell wall-associated genes and the observed mutations in some cell wall-associated genes, particularly RLM1, a gene that encodes a transcription factor responsible for the activation of the MPK1 mitogen-activated protein kinase pathway, and the maintenance of cell wall integrity. Some of the missense mutations of 2E were found in vesicle protein sorting and vacuole biogenesis (VPS45, PEP5), and endocytosis (ENT1, YAP1802, MYO5, AKR2) genes. Also AAC1 that encodes a vesicular transport protein was upregulated in 2E. As a result, the silver-resistant evolved strain 2E kept higher levels of silver in/on itself, compared to the reference strain, according to Flame Atomic Absorption Spectrometry (F-AAS) analysis results, that possibly made a significant contribution to its silver resistance. This study was about selecting and characterizing a silver-resistant mutant of S.cerevisiae at a molecular level. The possible reasons for the silver resistance in yeast cell were discussed, regarding molecular and functional factors. Some potentially important genes and pathways for the silver resistance mechanism have been identified in this study, which have been discussed, based on the previous studies found in the literature about silver response. Despite these major findings, further investigations are still needed to fully enlighten the complex molecular mechanisms of silver resistance, regarding copper resistance, oxidative stress, cell wall integrity, aerobic metabolism and vesicular transport.
ÖgeInverse metabolic engineering and molecular characterization of caffeine-resistant saccharomcyes cerevisiae(Fen Bilimleri Enstitüsü, 2020)Caffeine, a natural purine alkaloid, is highly consumed worldwide as an ingredient of some beverages like coffee, tea and soft drinks. Although there are many reports on caffeine effects in many organisms like yeast, pleiotropic effects of caffeine, and molecular mechanisms of caffeine resistance and response are largely unknown.In this study, highly caffeine-resistant Saccharomyces cerevisiae mutants were obtained for the first time by evolutionary engineering, an inverse metabolic engineering strategy, based on batch selection under gradually increasing caffeine stress, with and without applying any mutagen to the initial population before selection. The selection process was initiated at 7.5 mM caffeine concentration, and continued until 50 mM, for 48 populations. Genetically stable, caffeine-resistant mutant strains were isolated from the final populations of the selection, where they resisted as high as 50 mM caffeine, a concentration that has not been documented for S. cerevisiae so far. Among those mutants, three strains Caf905-2, Caf906-11, and Caf906-12) had the highest caffeine resistance, and they were also cross-resistant to a variety of stress conditions like coniferyl aldehyde, antimycin A, and rapamycin. One of the caffeine-hyperresistant strains (Caf905-2) was characterized at physiological, transcriptomic and genomic levels. Caf905-2 did not have a reduction of growth under 10 mM caffeine stress, where its maximum specific growth rate (µmax) was nearly three-fold of that of the reference strain. Expectedly, Caf905-2 entered the stationary phase at 12 h, but the reference strain at 30 h. Biomass levels of the two strains showed an increase under 10 mM caffeine stress, compared to the absence of caffeine. Also, ethanol yield decreased in the reference strain under 10 mM caffeine stress, but not in Caf905-2. Similarly, glycerol yield decreased in Caf905-2 under caffeine stress, but not in the reference strain. The reference strain also accumulated more intracellular trehalose than Caf905-2, particularly under caffeine stress condition. The resistance of Caf905-2 against lyticase, a β-1,3-glucan-degrading enzyme, was higher than that of the reference strain, under both caffeine stress and nonstress conditions. DNA microarray analysis identified a total of 745 overexpressed and 741 repressed genes with statistically changes of gene expression levels (corrected p < 0.05) encompassing at least 2-fold alteration in Caf905-2. Functional categorization of transcriptomic analysis results of Caf905-2 suggested that the genes implicated in energy and protein fate pathways, oxygen and radical detoxification, and drug/toxin transport associated with pleiotropic drug resistance were stimulated, whereas the genes implicated in transcription, protein synthesis, cellular transport pathways, and nucleotide metabolism were repressed in the nonstress condition. Whole genome re-sequencing analysis results unearthed only three single nucleotide variations, distributed in three different genes; PDR1, PDR5, and RIM8 in Caf905-2, relative to the reference strain. The exact roles of these genes in caffeine response and resistance in yeast should be investigated in detail in future studies.
ÖgeDesign of a bioactive scaffold system for hard tissue engineering(Institute of Science and Technology, 2013)Nanofibrous double-layer matrices were prepared by electrospinning technique with the bottom layer formed from PCL (poly--caprolactone) / PLLA (poly-l-lactic acid) blend nanofibers and the upper layer from PCL/Gelatin blend nanofibers. Gelatin microspheres were incorporated physically in the middle of these two layers for controlled growth factor delivery. This sandwich system prevented microsphere leakage from the scaffold. Bead-free nanofibers with uniform morphology could be obtained by 10% w/v concentrations of PCL/PLLA and PCL/Gelatin solutions. Microspheres prepared by 500-rpm stirring rate and cross-linked with 7.5% glutaraldehyde solution were chosen after in vitro release studies. The optimized conditions were used to prepare fibroblast growth factor-2 (FGF-2) loaded microspheres. Cell culture studies with FGF-2 loaded microspheres showed that the growth factor could be actively loaded into the system and enhanced the cell attachment and proliferation. To test the effect of gelatin on cell adhesion, cell culture was performed with PCL/PLLA matrices with or without PCL/Gelatin layer. Cell attachment was significantly higher when PLLA replaced by gelatin. To investigate cell differentiation on the designed scaffold system adipose tissue derived stem cells (ADMSCs) were used and microspheres within the scaffolds were loaded with bone morphogenic protein2 (BMP-2). The effect of scaffolding on differentiation of ADMSCs towards osteogenic lineage was evaluated by various markers. According to the results, all experimental samples were biocompatible and supported the proliferation and differentiation of ADMSCs.
ÖgeEvolutionary engineering and molecular characterization of freeze-thaw resistant saccharomyces cerevisiae(Fen Bilimleri Enstitüsü, 2013)Saccharomyces cerevisiae içki ve fırıncılık endüstrisinde yaygın bir şekilde kullanılan endüstriyel bir mikroorganizmadır. İyi karakterize edilmiş bir mikroorganizmadır ve aynı zamanda temel ve biyoteknolojik araştırmalar için önemlidir. Endüstriyel uygulamalarda, donma-erime stresi özellikle donmuş hamur prosesinde genel bir stres çeşididir. S. cerevisiae? de donma-erime stres uygulamaları, hücrelerin donma-erime stresinin ölümcül etkileri ile başa çıkabilmeleri için çok yönlü çapraz-direnç mekanizmasını uyarır. Çok yönlü stres direnci ve donma-erime toleransı elde etmek için ekstra genetik mekanizmaların donma-erime stresi varlığında uyarıldığı ve aktif oldukları düşünülmektedir. S. cerevisiae? de donma-erime stresi ile ilgili birçok çalışma bulunmaktadır, fakat donma-erime toleransının arkasında yer alan moleküler mekanizmalar hala belirsizliğini korumaktadır. Evrimsel mühendislik, donma-erime stresine direnç gibi, özel ve istenilen fenotiplerdeki mutant suşların elde edilmesinde kullanılan ?doğal? bir prosestir. Genel olarak, evrimsel mühendislik dört ana basamaktan oluşmaktadır. Bu aşamalar; genetik olarak karışık hücre populasyonunun kimyasal/fiziksel mutasyon ile elde edilmesi, istenilen özellikteki geliştirilmiş suşun elde edilmesi için tekrarlayan sikluslarda seleksiyon baskısının uygulanması, elde edilen suşun performansının analizi ve bir sonraki hedefin dizaynı. Sonuç olarak, endüstriyel uygulamalarda, yüksek derecede donma-erime stresine dirençli, mayanın kendi genomu dışında yabancı herhangibir gen içermeyen, geliştirilmiş ekmek maya hücrelerinin kullanımı, daha güvenli ve kabul edilebilirdir. Böylece, bu yöntem, donma-erime stres direnci ile sonuçlanan yüksek derecede regüle edilmiş genomu ile ekmek mayasının endüstride kullanımının güvenli bir yoludur.