LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji-Yüksek Lisans

Bu koleksiyon için kalıcı URI

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

Gözat

Yazar "Erdoğdu, Nazlı Dilara" ile LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji-Yüksek Lisans'a göz atma
  • Öge
    The production and pegylation of recombinant human granulocyte colony-stimulating factor produced in e. coli
    (Graduate School, 2023-07-14) Erdoğdu, Nazlı Dilara ; Doğanay Dinler, Gizem ; 521201120 ; Molecular Biology-Genetic & Biotechnology
    This thesis is composed of two distinct subjects separated by two chapters. The first chapter focuses on the "Production, purification, and pegylation of G-CSF". The granulocyte colony-stimulating factor (G-CSF) is a cytokine and has a role in the maturation, differentiation, and migration of white blood cells. The G-CSF protein is an 18-kDa protein and has a theoretical pI of 5.6. 5 cysteine residues form 2 disulfide bonds and one free cysteine locates at the 18th position. White blood cells are one of the major systems used against pathogens. The absence or low abundance of white blood cells results in a condition called neutropenia. Neutropenia patients require admission of G-CSF in its drug form, which is called filgrastim. Filgrastim is a recombinant therapeutic protein, and due to its low molecular weight, its elimination through the kidneys causes a short half-life in blood circulation. To solve the short half-life problem, a chemically modified form of G-CSF has been created. The N-terminus of the protein is modified with a 20 kDa polyethylene glycol (PEG) that increases the molecular weight by two-fold. The chemical addition of a PEG molecule is called pegylation, and several types of pegylation vary in terms of the molecular weight or the structure of PEG, or the target of the pegylation. The Food and Drug Administration (FDA) approved N-terminus pegylated filgrastim, which is called Pegfilgrastim and it is the first FDA-approved pegylated drug. To add the PEG to the N-terminus, the methoxy-PEG aldehyde and the chemical reaction called reductive amination are terminated with the addition of a reducing agent. The common pegylation method involves the addition of a reducing agent, sodium cyanoborohydride that forms the toxic cyanide as a byproduct. This study aims to find a high-yield protein production strategy from bacterial cells and to find an alternative to sodium borohydride. First of all, to increase the yield of protein production in E. coli BL21, the existing method in our lab was optimized. The protein is produced under the Lac operon promoter and the production is induced with IPTG. At 37 °C, the protein is produced at the inclusion body and needs to be solubilized and refolded. 8 M urea seems to be inefficient to solubilize the protein, therefore an alkali buffer was also tried and compared to the former method. Then, the bacteria incubated at 17 °C produced the protein in soluble form. That method of soluble protein production has been tried. To overcome the instability, the 18th cysteine was substituted to a serine residue with site-directed mutagenesis. Even though the alkali buffer has a higher yield of solubilization, the process is not suitable overall due to the instability of the G-CSF at pH values over 5.5. The soluble protein method was inconvenient, as this method yields more impurities in the elution. The mutation increased the stability of the protein in the dialysis steps; however, it was not enough to produce the protein with a higher yield. The second part was about pegylation of G-CSF. The substitution of sodium cyanoborohydride with sodium borohydride was not successful, as the latter is not stable in aqueous solutions. As the optimized method was not suitable to produce G-CSF in an industrial scale, other methods such as adding a solubilizing tag or a tag to help purification which also increases the size of the protein can contribute to increasing the half-life and also the purification of the protein. Also, this thesis contributed to the literature by the trial of Sodium borohydride as a reducing agent in pegylation reaction and showed the inefficiency of the chemical due to its instability. The second chapter of the thesis is about the "Characterization of CHEK2 VUSes found in genetic screening of Turkish breast cancer patients". The CHEK2 gene is a tumor suppressor and encodes the protein serine-threonine kinase Checkpoint kinase 2 (CHK2). CHK2 protein consists of three functional domains; SQ/TQ cluster domain (SCD), forkhead-associated (FHA) domain, and the kinase domain. The SCD is a target for CHK2 activator proteins. The FHA domain has a role in the dimerization and activation of the CHK2 monomers by trans-activation through further phosphorylation. The kinase domain is where CHK2 binds to its downstream targets and phosphorylates them. CHK2 is involved in a double-strand DNA break repair mechanism. Firstly, ATM is activated by double-strand break recognition proteins, and it phosphorylates CHK2 at the 68th threonine. CHK2 then phosphorylates and activates the pathways of cell cycle delay, DNA repair, and apoptosis. There are variants of CHEK2 that cause non-functional proteins and are therefore related to Li-Fraumeni syndrome or several cancer types. The pathogenic variants of CHEK2 are known to be associated with breast, colon, kidney, and prostate cancers. However, some variants are not identified as either pathogenic or benign. These variants are called Variants of Unknown Significance (VUS). In the previous study, Akcay and colleagues found that CHEK2 is the most VUS-carrying gene among 25 other cancer-related genes in Turkish breast cancer patients. To be able to classify the variants of unknown significance in vitro, the optimization of CHKk2 production should be performed. This study aimed to produce and purify recombinant wild-type and mutant CHK2 proteins in E. coli. First of all, the gene is cloned into the expression vector pET30a with 6xHis and 3xFLAG tags. The E. coli strain BL21 was chosen to produce the protein, and the production is optimized. 17 °C was selected as the incubation temperature after induction with IPTG. The soluble fraction after lysis is used to purify CHK2 with immobilized metal affinity chromatography (IMAC). After the purification is optimized, circular dichroism spectroscopy is used to identify the secondary structure of the protein via far-UV measurement. Between the 6xHis tag and the 3xFLAG tag, the TEV enzyme restriction site was additionally provided to eliminate contaminants via a second IMAC after TEV digestion. However, this was not used because the protein is relatively pure and the majority of the contaminants come from cleaved CHK2 protein. The secondary structure of the wild-type and mutant proteins revealed that the purified protein was correctly folded as the CD curve matches an alpha-helical protein. The in vitro studies using the recombinant CHK2 may resolve the effect of the mutations on structure and function of the protein.