تولید و بررسی ویژگیهای آنزیم نوترکیب ریبونوکلئاز پانکراس گوسفند

همان‌طور‌که تحقیقات گوناگون نشان داده‌اند، در سال‌های اخیر، میزان مرگ‌ومیر براثرِ سرطان رشد چشمگیری داشته است؛ به‌همین‌دلیل است که محققان درپیِ تولید داروهای جدید، به‌ویژه داروهایی با اثرات جانبی کمتر، هستند. برهمین‌اساس، آنزیم ریبونوکلئاز پانکراس گوسفند نیز یکی از بهترین اورتولوگ‌ها برای آنزیم ریبونوکلئاز پانکراس گاو است که آن را در القای مرگ سلول‌های سرطانی به‌کار می‌گیرند. آنزیم گوسفندی تنها در 4 اسیدآمینۀ a19s، k37q، v46f و n103e با آنزیم گاوی تفاوت دارد. در مطالعۀ پیش‌رو، ابتدا شکل سه‌بعدی آنزیم ریبونوکلئاز پانکراس گوسفند پیش‌بینی و سپس از‌نظر پایداری بررسی شد. درادامه، مشخصات فیزیکوشیمیایی این آنزیم با برنامه پرات پارام نیز مورد بررسی قرار گرفت.  همچنین، با استفاده ازروش‌های بیوانفورماتیکی شبیه‌سازی و با‌توجه ‌به rmsf، rmsd و gyration analyses میزان پایداری پروتئین نیز پیش‌بینی گردید. مطالعات آزمایشگاهی نیز از ‌قبیل بررسی فرار این آنزیم از ri، میزان مقاومت آن درمقابل پپسین، غلظت پروتئین تولیدی، قابلیت القای مرگ سلولی در سلول‌های سرطانی و نیز میزان فعالیت این آنزیم، انجام گرفت. نتایج این تحقیق نشان می‌دهد که آنزیم مدنظر پس از 22 ساعت انکوبه در مجاورت پپسین تا 90% دست‌نخورده باقی می‌مانَد. میزان غلظت پروتئین تولیدی نیز mg/ml 4.78 بود که با استفاده از روش برادفورد تخمین زده شد. در این مطالعه فعالیت آنزیم ریبونوکلئاز طبیعی پانکراس گوسفند 558±22 u/mol برآورد شد. همان‌طورکه در تحقیقات گذشته نشان داده شده است، انواعِ rnasea گاو و گوسفند نمی‌توانند وارد سلول شوند و درنتیجه هیچ‌گونه سمیت سلولی نشان نمی‌دهند، ولی در این مطالعه با استفاده از لیپوفکتامین، این آنزیم‌ها وارد سلول شدند و مرگ سلولی را به سلول‌های سرطانی hella القا کردند. میزان زنده‌مانی سلول‌ها نیز به‌روش mtt assay  اندازه‌گیری شد. براساس این تحقیق، مشخص شد که آنزیم ریبونوکلئاز پانکراس گوسفندی فعالیت آنزیمی و توکسیتی مشابهی با آنزیم ریبونوکلئاز پانکراس گاوی دارد و برای استفاده به‌عنوان توکسین در تولید ایمونوتوکسین‌ها جانشین مناسبی است.

Production of ovine Pancreatic Ribonuclease and investigation of enzyme characteristics

Introduction Obviously, the recent decades strategy in cancer therapy, anticancer drug discovery and drug improvement is to characterize, distinguish and validate the most promising cancerrelated molecular targets to which new drugs can be designed. The unique features of the pancreatic RNase (RNase A) such as high activity, stability, lack of a cofactor, and small molecular size have made it the most popular enzyme in the ribonuclease family. Specifically, RNase A is involved in endonucleolytic cleavage of 3’phosphomononucleotides and 3’ phosphooligonucleotides ending in CP or UP with 2’, 3’ cyclic phosphate intermediates. RNase A was purified from the Bovidae family including bovine, ovine, bison, eland, goat and gnu. Although phylogenetic analyses of RNase A revealed high similarity among members of the Bovidae family, some functional mutations were also found.Several studies showed that the RNA hydrolyzing action of ribonuclease is able to induce apoptosis and cell death in cancer cells, independently. This effect could be enhanced thousands of times when ribonuclease is linked to antibodies. These enzymes show potent cytotoxic activity on cell internalization but do not show sensible immunogenicity or nonspecific toxicity toward normal cells. Ovine pancreatic ribonuclease enzyme is a member of super family RNase A, it can be a good candidate as a toxin for designing new drugs. The objective of this study was to produce ovine pancreatic ribonuclease enzyme in E. coli and characterize its activity.Materials and methods All structures needed for this study were downloaded from the Protein Data Bank (PDB) website (http://www.rcsb.org/). PDB files (accession numbers: 10.2210 /pdb1YV6/pdb and 10.2210/pdb3SNF/pdb) related to natural Bovine Pancreatic RNase were selected. Gene synthesis and production of recombinant protein were conducted by using the pelB signal sequence at the beginning of the structure for periplasmic protein production. The native ovine RNase and bovine RNase A were optimized for E. coli host by GenRay codon optimization service and sent to GenRay Biotechnology (Shanghai, China) for synthesis. The target genes in pGH vector were subcloned in pUC19 and then cloned into the pET21b (+) vector between XbaI and HindIII sites. After transformation, E. coli cells containing recombinant pET21b (+) were cultivated in LB broth medium containing ampicillin. To extract the proteins, osmotic shock methods were applied. After that Qsepharose chromatography was used to extract the target protein. Finally, Bradford analysis was used to determine the protein concentration. The ribonucleolytic activity of the recombinant native ovine RNase A was compared with native bovine RNase A following Tripathy et al (2013) method. To investigate the antitumor activity of recombinant proteins, HeLa cells were prepared for seeding in a 96well flask and incubated at 37°C in 5% CO2 for 72 h.Results and discussion The production of native ovine and bovine RNase A was confirmed by SDS PAGE. Protein purification was successfully performed using osmotic shock in the QSepharose column. Although our findings confirmed protein expression, no detectable proteins other than RNase A was observed in the LB medium, indicating that almost all the proteins were expressed either inside the bacterial cell or secreted into the periplasmic area. According to the Bradford analysis, the concentration of the recombinant proteins extracted was 4.78 ovine RNase A. Based on our results, it was shown that the ribonucleolytic activity of native ovine RNase A was 558±22. The results showed that RNase A exhibited resistance to pepsin degradation during the whole incubation process (22 h), in the course of which 90% of RNase A remained undamaged. To determine the cytotoxic effect of ovine RNase on the HeLa cell line, MTT assay was done following incubation with ovine RNase A. The commercial RNase A was used as control. The results showed that ovine RNase A and bovine RNase A had no cytotoxic effect on HeLa cells. When RNase treatment was done by the lipofectamine 3000 (Thermo, USA), the cytotoxicity effect was observed. Several studies have shown that some ribonucleases such as onconase, bovine seminal ribonuclease and bovine pancreatic ribonuclease have a great promise as cancer immunotherapeutic agents and cause a significant reduction in the protein synthesis of tumor cells after internalization into cytosol. Conclusion Our findings demonstrate that ovine RNase similar to bovine RNase has a great potential for use in drug design industry. We revealed that the native ovine RNase A was more stable than the native bovine RNase. In future work, we intend to fuse the engineered ovineRNase A to dedicated recombinant antibodies for cancer therapy and investigation of engineered immunoribonuclease potency and cell killing effects as a fusion protein.