LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji-Yüksek Lisans

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  • 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.