Bacillus subtilis-derived endo xylene Rana proposed signal sequence of the method for producing foreign proteins by using recombinant plasmid containing the gene and him

摘要

PURPOSE: Recombinant plasmids having secretion signal sequence of endoxylanase from Bacillus are provided which express and secret foreign protein in transformed E. coli therewith. CONSTITUTION: A secretion signal sequence of endoxylanase fragment from plasmid, pKJX4, containing endoxylase gene from Bacillus is amplified by PCR. Recombinant plasmid, pJS101, is constructed by cloning PCR fragments into plasmid, pTrc99A, having strong trc promoter. Plasmid pJS101 contains signal sequence of endoxylanase and endoxylanase joined with PstI site. 3' end of endoxylanase has restriction sites of BamHI, XbaI, SalI, PstI and HindIII for the cloning of foreign protein. Plasmid pJS101DeltaP is constructed by removing PstI site of 3' end of endoxylanase gene for the easy introduction of foreign protein. T7 promoter containing vector, pJS301, is constructed by joining of NdeI and BamHI digested fragment from pJS101DeltaP and pET21c. Induction experiment with IPTG shows that the yield of secreted protein is at least 6% of total protein. Alkaline phosphatase is over expressed and constitutes 6.9% of total protein.