High level secretory expression of natural N-terminal rBPTI with human serum albumin signal peptide in Pichia pastoris

Objective To explore the method of secretory expression of the natural N-terminal rBPTI with high-level in Pichia pastoris.Methods Human serum albumin signal peptide (hsasp) and bpti genes were ligated and the eukaryon expression plasmid pPICZ/hsasp-bpti was constructed.The recombinant plasmid was transformed into the Pichia pastoris(X-33)via electroporation.The transforming positive strains were screened by PCR,SDS-PAGE and trypsin inhibition experiment.Results The rBPTI was expressed and secreted in X-33.SDS-PAGE and MS showed that the relative molecular mass of rBPTI were respectively 6 500 and 6 508.Sequence analysis of amino acid proved that 15 amino acids at amido-rBPTI were identical with that of natural BPTI.Trypsin inhibition experiment showed that Ki value of rBPTI[(2.6±0.1)×10-9]was identical with that of natural material.Conclusion rBPTI with natural N-terminal sequence is successfully expressed in Pichia pastoris with hsasp,and the expression level of rBPTI reaches at 200 mg·L-1.