LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji-Yüksek Lisans
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Yazar "Doğanay Dinler, Gizem" ile LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji-Yüksek Lisans'a göz atma
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ÖgeCharacterization of BAG-1S/C-raf interaction targeting peptide(Graduate School, 2022-07-25) Çebi, Ecenur ; Doğanay Dinler, Gizem ; 521201110 ; Molecular Biology-Genetics and BiotechnologyERK/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.
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ÖgeExpression, purification and characterization of high-fidelity DNA polymerase(Graduate School, 2022) Türk, Kübra ; Doğanay Dinler, Gizem ; 783309 ; Molecular Biology-Genetics and Biotechnology ProgrammeDNA 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.
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ÖgeExpression, purification, and characterization of recombinant human IL-2(Graduate School, 2022-01-18) Akgün, Buse ; Doğanay Dinler, Gizem ; 521181103 ; Molecular Biology – Genetics and BiotechnologyCytokines, 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.
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ÖgeFunctional 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 & BiotechnologyLynch 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.
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ÖgeThe 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 & BiotechnologyThis 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.