| Snake venom thrombin-like enzymes(SVTLEs),which belong to the serine proteases of trypsin family,mainly distributed in the venom of Viperidae crotalinae.In vitro,SVTLEs can directly act on specific parts of the fibrinogen to cleave the peptide bond between Arg and Gly residues,releasing of fibrin peptides and leading to fibrin monomer polymerized and formed clots.But they can't activate cogulation factors in vivo because the degrading products of fibrinogen can't form side cross-linked chains,which is easy to be cleared by normal reticuloendothelial system or normal fibrinolysis,thus showing fibrinolytic and anticoagulant effects.Clinically,SVTLEs have become effective drugs for the prevention of thromboembolic disease.However,it is difficult to gurantee the production due to the limited resources of snake venom and high cost of purfication.Therefore,genetic engineering is one of the best alternatives to meet the demand.Pichia pastoris is one of the eukaryotic expression system,which has been successfully expressed a variety of proteins and many of SVTLEs have been expressed now,but there were less genetic engineering studies about SVTLEs of Gloydius intermedius.Therefore,in this study we used two SVTLEs genes from the venom gland cDNA library of Gloydius intermedius as materials,optimized the codons and mutated two Kex sites,and then expressed in Pichia pastoris and biological activities were characterized.Through this research,we expected to lay the foundation for further study of SVTLEs of Gloydius intermedius.The main contents of this research as follows:1.Two serine protease genes(Intobinl and Intobin2),which came from cDNA library of Gloydius intermedius venom glands,were subjected to bioinformatics analysis.2.The codons of Intobinl and Intobin2 genes were optimized by the codon usage bias of Pichia pastoris,then the optimized genes were artificially synthesized and the mutant Intobinl and Intobin2 gene without Kex2 sites were designated as mIntobinl and mIntobin2.3.The optimized genes were inserted into the secreted expression vector pPIC9K to construct the recombinant vector of pPIC9K/mIntobinl and pPIC9K/mIntobin2.4.Recombinant plasmids were transformed into Pichia pastoris GS115 by electroporation and the positive recombinant strains were screened with different concentrations of G418 and MD plates.Yeast genome extraction kit was used to extract the genome DNA of GS115/pPIC9K/mIntobinl and GS115/pPIC9K/mIntobin2,PCR was used to verify its phenotype.And the correct clones were capable of expressing mIntobinland mIntobin2 in culture medium.At last,the fermentation supernatants were detected by SDS-PAGE and Western-blot.5.The fermentation conditions were optimized from five aspects:culture time,inducing methanal concentration,temperature,pH and different antioxidiants.mIntobinl and mIntobin2 proteins were purified by Ni-NTA column.6.The supernatant concentration and production of protein were measured by bradford method,and the purity of protein was analysed by Gel-Pro.7.The Amide hydrolase activity was detected with the simulation substrate BapNA.The fibrinolytic activity was assayed on the fibrin plates;The fibrinogen hydrolysis activity was analyzed by SDS-PAGE.The results were as following:1.The result of bioinformatics analysis showed that intobinl has an open reading frame of 789 bp,encoding 262 amino acids with the 18N-terminal amino acids as signal.the molecular weight of mature peptide is 26.9 kD and the theoretical isoelectric point is 6.76.the potential N-glycosylation sites are 105 and 124.the amino acid sequence of Intobinl has a high homology with Pallabin-2(Gloydius halys).Intobin2 has an open reading frame of 783 bp,encoding 260 amino acids with the 18N-terminal amino acids as signal.the molecular weight of the mature peptide is 26.55 kD and the theoretical isoelectric point is 8.43;the potential N-glycosylation sites are 123 and 124;the amino acid sequence of Intobin2 has a high homology with Gloshedobin(Gloydius Shedaoensis).2.The optimized results showed the amino acid sequence of Intobinl and Intobin2 remain the same between the optimized genes and original genes.The optimized genes were replaced 18 amino acids and 17 amino acids in Intobinl and Intobin2.And the adjusted G+C content of Intobin1 and Intobin2 were 42.89%and 43.71%,respectively.The Arg of Intobinl and Intobin2 was mutated to Lys in order to prevent the Kex2 on protein degradation.3.The PCR and sequencing result showed that the recombinant plasmids pPIC9K/mIntobin1 and pPIC9K/mIntobin2 were successfully reconstructed.4.Linearized expression vectors pPIC9K/mIntobinl and pPIC9K/mIntobin2 were introduced into the Pichia pastor is GS115.Screening under different concentrations of G418,high resistant transformants were obtained.The results of MM and MD plates showed that the methanol utilization types were Mut+;the result of PCR showed that the target genes have been successfully integrated into the genome of GS115.SDS-PAGE analysis showed the recombinant proteins have expressed,with the bands molecular about 35.0 kDa.5.The optimized fermentation conditions of GS115/pPIC9K/mIntobinl showed as follows:120 h,the methnol concentration of 1%,30?,pH 6.0,adding melatonin and Vc could improve the expression of GS115/pPIC9K/mIntobinl.The optimized fermentation conditions of GS115/pPIC9K/mIntobin2 showed as follows:96 h,the methnol concentration of 1%,28?,pH 6.0,adding melatonin and Vc could also improve the expression of GS115/pPIC9K/mIntobin2.The result of purification showed mIntobin1 and mIntobin2 had the single band with molecular weight about 35.0 kDa.6.Induction under the optimal condition,the expression of the supernatant of GS115/pPIC9K/mIntobinl and GS115/pPIC9K/mIntobin2 reached a level of 265 ?g/ml and 245 ?g/ml,respectively.Compared with the initial induction,the expression under optimized condition increased by 48.5%and 47.3%.The concentration of purified protein mIntobin1 and mIntobin2 were 75 ?g/ml and 60 ?g/ml,and there were no obvious change of production compared with intobin and intobin2 which were not mutated.Gel-Pro analysis found the purity of mlntobinl and mIntobin2 reached a level of 80%and 70%,respectively.7.The biological activities showed the purified mlntobinl and mIntobin2 had no amide hydrolase activities and fibrinolytic activities,but had fibrinogen hydrolytic activity.After incubation of bovini fibrinogen with mlntobinl,mIntobin1 can cleave both Aa chain and B? chain of bovini fibrinogen slowly with time going on;whereas mIntobin2 can cleave B? chain of bovini fibrinogen,degradation of the B? chains was observed when time was 30 mins.However,mIntobin1 and mIntobin2 can not cleave y chain of bovini fibrinogen.Conclusion:This work has established an effect way to expression mIntobinl and mIntobin2 and laid the foundation for further study of the gene of mIntobin1 and mIntobin2. |
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