| Thrombotic diseases,such as arterial-venous thrombosis,myocardial and cerebral infarction,represent serious health concerns.Thrombolysis is considered the most effective treatment for these kinds of diseases.Thrombin-like enzymes and plasmin represent venom-based anti-thrombotic drugs currently in clinical use.Snake venom plasminogen activator displays a long circulating half-life,high selectivity and converting human Glu-plasminogen into active plasmin.Hence,it has become a hot spot in the development of noval thrombolytic agents.The snake venom of Gloydius intermedius is a rich source of serine proteases,which has important value in the fields of basic research and medicine.However,due to the limitation of snake venom resource and difficulty to obtain enough amount for clinical use,in the present study,with two serine protease genes(designate SP20638 and SP46086)as material,we aim to express these two serine proteases in Pichia pastoris system,so as to obtain recombinant proteins and characterize their activities.The main content can be briefly summarized as follows:1.Using bioinformatics softwares to analyze SP20638,the results showed that the open reading frame of SP20638 is 777 bp,encoding 258 amino acids.The protein encoded by the gene is a plasminogen activator,belongs to the PA subtype.The N-terminal 24 amino acids is signal peptide(1-18)and propeptide(19-24),so the mature peptide contains 234 amino acids(25-258).The potential N-glycosylation site is Asn44.Three residues(His65,Asp110,Ser204)form the active sites.The twelve conserved Cys residuces are presumably to form six disulfide bonds(Cys31-Cys163,Cys55-Cys66,Cys98-Cys256,Cys142-Cys210,Cys174-Cysl89,Cys200-Cys225),with the theoretical molecular weight and isoelectric point as 25.4 KDa and 5.30.The open reading frame of SP46086 is 783 bp,encoding 260 amino acids.The protein encoded by the gene is a thrombin-like enzyme with kinin releasing activity,belongs to the KN subtype.The N-terminal 24 amino acids is signal peptide(1-18)and propeptide(19-24),so the mature peptide contains 236 amino acids(25-260).The potential N-glycosylation sites are Asn123 and Asn124.Three residues(His67,Asp112,Ser206)form the active sites.The twelve conserved Cys residuces are presumably to form six disulfide bonds(Cys31-Cys165,Cys52-Cys68,Cysl00-Cys258,Cys144-Cys212,Cys176-Cys191,Cys202-Cys227),with the theoretical molecular weight and isoelectric point as 25.9 KDa and 8.44.2.The genes SP2063 8 and SP46086 were optimized by partly replacing the P.pastor is preferred codons(synonymous codons).Then the optimized genes were synthetised by Sangon Biotech.The results showed below,after codon optimization,the free energy of the folding mRNA was increased from ?G=-237.20 kcal/mol to?G=-173.30 kcal/mol compared with the native SP20638 gene,the codons of 15 amino acids were replaced by their synonymous codons,a toal of 85 loci,the G+C content was decreased from 52%to 39%,the optimized SP20638 gene is 729 bp.The free energy of folding mRNA was also increased from AG=-224.20 kcal/mol to ?G=-173.80 kcal/mol compared with the native SP46086 gene,the codons of 17 amino acids were replaced by their synonymous codons,a total of 99 loci,the G+C content was decreased from 48%to 38%,the optimized SP46086 gene is 735 bp.The optimized SP20638 gene and SP46086 gene were named GI-PA1 and Intobin2,respectively.3.To construct the expression vectors,the optimized gene sequences were ligated to the corresponding sites of the expression vector pPIC9K by double digestion using SnaB I and Not I.Then the constructed plasmids were transformed into competent E.coli Top 10 strain,which was spread on LB agar plates with 100 ?g/mL kanamycin,and incubated overnight.The expression plasmids were verified by both SnaB I/Not Idigestion and PCR,then sent to Sangon Biotech for sequencing.The results showed that the recombinant plasmids pPIC9K/GI-PA1 and pPIC9K/Intobin2 were successfully constructed.4.P.pastoris strains(GS115 and SMD1168)were grown in yeast extract peptonedextrose(YPD)medium and prepared for transformation,according to the manufacturer's instructions.After linearization of the corresponding recombinant plasmids with Sac I,the P.pastoris strains were transformed using electroporation method(GI-PA1 gene was transformed into GS115 strain,Intobin2 gene was transformed into SMD1168 strain),the electroporation pulse was carried out at 1500 V,25 ?F,200 ?.After incubation at 30 ? for 2-3 days,clones were picked and spotted onto YPD agar plates containing 1,2,3 and 4 mg/mL G418(shanghai,China)to screen for putative multiple copies recombinants.Then the His+ transformants were replica-plated onto MM plates and MD plates to determine methanol-utilizing phenotypes.The transformants were grown at 30 ? for 3-4 days.The Mut+ phenotypes were grown normally on both MM and MD whereas the Muts phenotypes were grown very slowly on MM plates.Single colonies were isolated for analysis of protein expression.The obtained transformants were confirmed by a genomic PCR assays using 5'AOX1 primer and 3'AOXl primer.Each of the five positive colonies were cultivated in BMGY and induction in BMMY,respectivly.The clone that showed the highest level of protein expression(as detected by western-blot)was used to determine the optimum condition for expression by fermentation.The results showed that linearized expression vectors pPIC9K/GI-PAl and pPIC9K/Intobin2 were respectively introduced into P.pastoris GS115 and SMD1168.Screening under different G418 concentrations,five high G418 resistant transformants GS115/pPIC9K/GI-PA1 and five high G418 resisitant transformants SMD1168/pPIC9K/Intobin2 were obtained.Screening with MM and MD plates,the methanol utilization type were identified as Mut+.With the yeast genom DNA,the PCR results showed that the target genes have been successfully integrated into the genome of P.pastoris.After fermentation as well as western-blot,GS115 transformant 2 and SMD1168 transformant 1 were chose as the expreesion strains.Whereafter,we named the two strains as GS115/GI-PA1 and SMD1168/Intobin2.5.The selected recombinant strains were cultured in BMGY media and subsequently induced in BMMY media at 28 ? according to the protocol mentioned above.Then the optimized induction time,pH,final methanol,temperature and the adding of different antioxidants were determined.The SDS-PAGE of recombinant proteins were performed to determine optimized expression condition.The results showed that,the optimal induction conditions of GS115/GI-PA1 were 96 h,pH 6,final methanol concentration 1.5%,25 ?,adding 10 mmol/L ascorbic acid.The optimal induction conditions of SMD1168/Intobin2 showed as follows:96 h,pH 5,final methanol concentration 0.5%,25 ?,without adding antioxidants.6.Induced by the optimal fermentation conditions for large-scale expression,the recombinant proteins were purified by Ni-NTA affinity chromatography column.The amidolytic activity,fibrinolytic activity and fibrinogenolytic activity were determined after purification.The results showed that,the fermentation supernatants were successfully purified by Ni-NTA affinity chromatography column.Biological activities assay showed that rGI-PA1 and rIntobin2 didn't hydrolysis synthesized substrate BApNA.None of the enzymes have the fibrinolytic activity.The fibrinogenolytic activity of rGI-PA1 showed that the Aa and B? chains of bovine fibrinogen were degraded at dose of 2 and 3 ?g,and rGI-PA1 presented a higher ability to degrade B?chain.rIntobin2 seems to preferentially degrade both Aa and B? chains at the dose of 1?g.None of the enzymes were able to degrade the y chain of fibrinogen.Conclusion:The serine protease genes GI-PA1 and Intobin2 have been successfully cloned into P.pastoris GS115 and SMD1168.We obtain the recombinant strains GS115/GI-PA1 and SMD1168/Intobin2.The recombinant proteins have fibrinogenolytic activity.These works would lay the foundation for commercial production and application of Gloydius intermedius serine proteases. |
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