Abstract: To explore the method of secretory expression of the natural N-terminal rBPTI with high-level in Pichia pastoris. MethodsHuman 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 identi cal with that of natural material. ConclusionrBPTI 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.

Key words: human serum albumin signal peptide, Pichia pastoris