The Effective Expression Of Human Prorelaxin-2 Analogue In Pichia Pastoris

Relaxin is a reproductive hormone consisted of two short peptides,belongs to the insulin superfamily.Relaxin is important to reproductive tract and Cardiovacular.There are three relaxin genes including H1?H2 and H3,and the H2 gene is a dominantly functioning gene,existed as prorelaxin in the body.Prorelaxin is comprised of B-C-A domain configuration,and the C domain peptide is digested during the process of being mature relaxin.It is difficult to obtain pure relaxin from human tissues,so reseachers effort to produce recombinant relaxin.The frequent method was chemically synthesizing A chain and B chain,then vitro folding,but the disulfide bonds often mismatched,resulting to low yield.It is reported that recombinant relaxin has been produced in mammalian cells?E.coli and yeast et al,but the expression level and the biological action are both low.According to the primary structure of human relaxin H2 and Pichia pastoris preference codon usage,the recombinant prorelaxin(RH2)with artificial C domain petide was desiged and synthesized.With the yeast expressing vector pPICZaA and pGAPZaA transformed to Pichia pastoris GS115/SMD1168,the human recombinant prorelaxin H2 was received.After the artificial C domain peptide was digested,the active human recombinant relaxin H2 was obtained.1.According to the codon bias and the sequence of RH2,artificial C domain peptide inclcuding NGFNG were designed by Jcat.Then RH2 gene was inserted into the expressing vectors of pPICZaA-RH2?pGAPZaA-RH2 were synthesized.The results of PCR and enzyme digestion of plasmid proved that recombination was obtained.2.After linealization of the plasmids p PICZa A-RH2 with Sac I,it was inserted into the genome of host yeast GS115.After linealization of the plasmids pPICZaA-RH2 with Avr?,it was inserted into the genome of host yeast GS115 and SMD1168.The multiple clones were obtained by increasing concentrations of zeocin,then the results of PCR proves that the recombinat strains were obtained.3.pPICZaA-RH2-GS115 was cultured in BMMY under induction of methanol to optimize the expression time-conditions.Tricine-SDS-PAGE showed that the target protein was expressed in supernatant,and the highest expression level reached 451 ?g/mL at the 36 h,and the percentage of recombination reached 47.6 %.Western Blot showed specific reactogenicity of expressed protein.4.pGAPZaA-RH2-GS115 was cultured in YPD,and the optimization of expression time-conditions showed that the highest expression level reached 244 ?g/mL at the 36 h.The expression level of pGAPZaA-RH2-SMD1168 reached 470 ?g/mL,which was almost equal during 12 h to 72 h.Bioassay showed that the recombinant human relaxin was biologically active after purified and removed C peptide,and the purificaion reached 96%.Above all,yeast expression strains for Human Relaxin-2 analogue were constructed,and expressed in a secretory form in Pichia pastoris.After C domain peptide was digested by Hydroxyl ammonia.THP-1 exhibited a rapid and dose-dependent increase in the accumulation of cAMP by the recombinant relaxin we obtained.The successful construction and expression of the recombiant relaxin has laid a foundation for further study.