Journal
BIOLOGICAL & PHARMACEUTICAL BULLETIN
Volume 31, Issue 9, Pages 1680-1685Publisher
PHARMACEUTICAL SOC JAPAN
DOI: 10.1248/bpb.31.1680
Keywords
bovine pancreatic trypsin inhibitor; human serum albumin signal peptide; Pichia pastris; fermentation; purification
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Funding
- National High Technology Research and Development Program of China [2004AA205020]
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Bovine pancreatic trypsin inhibitor (BPTI) is a natural non-specific serine protease inhibitor and possesses the ability to inhibit trypsin, chymotrypsin, plasmin and plasma kallikrein. The expression of BPTI in Escherichia coli and other systems has been reported. However, the preparation of recombinant BPTI (rBPTI) with correct N-terminus in Pichia pastoris has not been successful. A previous study showed that the preBPTI with the prepro leader sequence of alpha mating factor (AMF) was not processed into natural BPTI in R pastoris. Now, we introduce a new method to prepare rBPTI, which carries a natural N-terminal amino acid residue, Arg-Pro-Asp, in I? pastoris using human serum albumin signal peptide corresponding to the pre sequence. The concentration of rBPTI in an 801 fermentor reached 900 mg/l. We also explored a rapid and simple purification protocol for rBPTI and the purity of rBPTI reached 95-98% as evaluated by SDS-PAGE analysis. The sequencing results showed that the sequence of N-terminal 15 amino acids of rBPTI was consistent with that of natural BPTI. The inhibitory activity of rBPTI against trypsin was the same as natural rBPTI and its K-i was 2.6 +/- 0.1 x 10(-9). The therapeutic effect of rBPTI on acute pancreatitis was identified in rats.
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