High expression of human relaxin-2 analogue in Pichia pastoris and activity of expressed product

Objective To highly express human relaxin-2 analogue(HR2)in Pichia pastoris,and determine the bioactivity of expressed product.Methods HR2 gene was synthesized by modification of codons,based on which recombinant plasmid pPICZaAHR2 was constructed and transformed to P.pastoris PGS115 for expression under induction of methanol.The expressed product was identified by Tricine-SDS-PAGE and Western blot,then purified by ultrafiltration and column chromatography,and determined for bioactivity after removal of synthetic C peptide.Results Restriction analysis and sequencing proved that recombinant plasmid pPICZαA-HR2 was constructed correctly.Tricine-SDS-PAGE showed that the expressed recombinant protein in a secretory form,with a relative molecular mass of about 6 500,contained 47.6%(451 μg/ml) of total somatic protein.Western blot showed good reactogenicity of the recombinant protein.The purified recombinant protein reached a purity of 96% and,after removal of C peptide,showed stimulating activity to THP-1 cells.Conclusion Recombinant HR2 was highly expressed in P.pastoris GS115,which showed a certain bioactivity.