High Expression Of Human Cu, Zn Superoside Dismutase In Pchia Pastoris And Pilot Production Procedure

SOD (superoxide dismutase, referred to as SOD) is a metal enzyme, which is an important organism defense enzyme to oxidative damage, widely distributed in aerobic organisms, oxygen-resistant organisms and some anaerobic organisms. SOD is known to be divided into thee categories that Fe-SOD, Mn-SOD and Cu, Zn-SOD.SOD could catalyze superoxide radical dismutation to hydrogen peroxide and oxygen. This enzyme in protecting the body from free radicals and superoxide generated by reactive oxygen species during oxidative damage plays a crucial role. At present, SOD has been widely used in the cosmetics, health food additive and so on. Zn-SOD was higher than the stability of Mn-SOD, and in mammals were much higher than in Mn-SOD, so many scholars pay more attention to Cu, Zn-SOD in clinical application.SOD in domestic market mostly though animal blood or plant extracts, therefore the use of effects and immunogenicity there was insufficient, and the body's tolerance of this mutant protein still need further study; In the process of extraction, easily occur the pathogenic microorganisms contamination and organic reagents residual; From 1997 the European Union banned the use of animals SOD, genetic engineering SOD, it overcomes the ills of SOD extracted from animals and plant. (mainly cross infection between viruses) and determines the bright future of the project.We chemically synthesized human Cu, Zn-SOD gene with the yeast bias codon, and cloned into integrated eukaryotic expression vectors pPIC9K, transformed into Pichia pastoris GS115 with different electroporation conditions, with the MD and MM plate for the table type screening, to obtain the positive transformants and achieve secretion of the expression induced by formaldehyde. We established pilot production procedure of fermentation and purification and liposomes to rhCu, Zn-SOD. It laid foundation for industrialization of rhCu and Zn-SOD.Pichia. Pastoris Gene expression system have been developed over ten years, has basically become a better exogenous gene expression system with easy-to-high-density fermentation, the expression of genes can be stably integrated in the host genome, can secrete protein and proper glycosylation, etc. At present, the US FDA has ability to evaluated the genetic engineering products from this system. The Cephelon from the system has been approved by FDA, so that the system is considered safe.We have successfully constructed 4 GS115/Cu,Zn-SOD transformants. Using Southern blot assay gene copy numbers of GS115/Cu, Zn-SOD transformants, study the stability of gene integration of thee strains which have higher copies of the Cu,Zn-SOD gene. The growth curves of the recombinant yeast entered the logarithmic growth phase after 16h, the stable growth phase after 24h. The Test determine the best time to induce. On one of the high-copy transformants were shake flask and pot culture condition optimization, Firstly, optimized the expression conditions at the flask level. The optimum cultivation conditions were:initial pH6.0,1.5% methanol concentration, harvest after 72 hours. Then, we optimized the expression conditions by 4 factors and 3 levels orthogonal design at the flask level. The optimal conditions were 1.5% methanol induce, pH6.0, temperature 30℃, harvest after72h. Obtain the optimal shaking induced 600 U/ml of activity of dimer protein, the molecular weight of 40KDa, monomer molecular weight of 20KDa, sugar glycation, both secretory expression of Cu, Zn-SOD antibody was specific reaction. TLCS electrophoresis analysis showed that the exocrine target protein accounted for 79.6% of the total protein. It was concluded that the stability of the enzyme:crude enzyme was stable in chloroform and acetone and ethanol in 60min, unstable on H2O2. In85℃within 15 minutes, and the range of pH4-10 were more stable.In this study, optimized the expression conditions in flask based on the orthogonal experiment, established the process of large fermentation tanks, that is the starting induced concentration of bacteria is A600200 in pH6.0 at temperature 25℃, fed methanol in 2ml/min flow rate, the activity of rhCu,Zn-SOD and the protein content of the expression reach the peak. Range analysis shows the concentration of methanol have the greatest impact on the induction of expression, and also shows that lower temperature were more conducive in large tank of high-density fermentation to the secreted expressed of protein. The results of tank fermentation showed rhCu, Zn-SOD protein was on average 66% of the total protein, was about 615mg/L, the activity were higher than 5000U/ml, the specificity activity were greater than 9×103U/mg on the optimum conditions, which were 8 times higher than in flask culture.The fermentation liquid of recombinant Cu,Zn-SOD in High-density tank was concentrated 5 (?)imes using ultrafiltration system, the activity of rhCu, Zn-SOD and protein recovery was up to 100%, the purification rate was up to 85%. Small number of experiments established the conditions for anion exchange charmatography, and explored the best conditions for salt and gel filtration. The purification results of five batches show that the rhCu, Zn-SOD concentrations of each batch were more than 1mg/ml. specificity activity were more than 6×103U/mg, rhCu. Zn-SOD protein activity and protein recovery were up to 70%, the purification was up to 95%. the quality can meet the requirement of the biological products of genetic engineering product inspection.This study established the methods of detecting rhCu, Zn-SOD protein content and rhCu, Zn-SOD activity.Reference to "Antibody Technology Experiment Guideline" prepared monoclonal antibodies and polyclonal antibodies for inspecting the specific of rhCu,Zn-SOD and established a double antibody sandwich ELISA detection methods (coated with polyclonal antibody and labeled with monoclonal antibodies), assembled rhCu,Zn-SOD detection kit for detection of expression of the fermentation broth. After indentified, the minimum detection limit was 18.75ng/ml, intra CV<10%, and inter CV<15%, the specificity was good. Overcome the bias of biochemical detection, truly reflected the levels of rhCu,Zn-SOD protein expression. It provides a reliable detection methods and experiments for the expression research. Pure rhCu, Zn-SOD meet the standards of genetic engineering products in Pharmacopoeia of the People's Republic of China (2005 Volume III). Non-reducing SDS-PAGE method and HPLC method for detecting rhSOD purity, Western blot method for detecting specific, reducing SDS-PAGE method for detecting the molecular weight, Coomassie brilliant blue staining and double antibody sandwich ELISA assay protein content, measured enzyme activity by activity kit, igoxigenin-labeled method for detecting residual DNA, ELISA assay for detecting residual protein, TAL for detecting the endotoxin, microorganism for detecting sterility test, isoelectric focusing for detecting Cu, Zn-SOD isoelectric point, the Edman degradation method for sequencing rhCu, Zn-SOD N-terminal amino acid. Examination results meet all indices of biological products.In order to better application, the pure enzyme was within liposomes prepared by rotary evaporation technique, combined with water renewal freeze-dried lipid film, orthogonal design to optimize fat plasmid modification rhCu, Zn-SOD conditions, analyzed affecting factors, prepared the rhCu, Zn-SOD liposomes under the optimal conditions, the average particle size is about 80nm. For rhCu,Zn-SOD liposome stability test, using gel filtration chomatography of free protein and rhCu,Zn-SOD liposomes to calculate encapsulation efficiency, which is up to 85%. The decrease of encapsulation efficiency was not obvious in 4℃and room temperature for 6 months. We can prepare large-scale stable rhCu, Zn-SOD liposomes. Pharmacokinetics showed that rhSOD liposomes significantly increased half-life in vivo, improved the bioavailability of rhCu, Zn-SOD that blood and liver tissue bioavailability was 195.94% and 134.86%, laid the foundation for clinical application.