Construction of the Escherichia coli-Bacillus subtilis shuttle vector pBE2R and identification of the critical residues involved in the autoprocessing of the propeptide of the alkaline protease

Abstract

A new Escherichia coli-Bacillus subtilis shuttle vector pBE2R was constructed to provide a convenient tool for the construction of the mutant library in the directed evolution of the alkaline protease. pBE2R was constructed by fusing P43 promoter with signal peptide as well as propeptide sequence of the apr E gene to the pBE2 vector. Alkaline protease can be expressed successfully in Bacillus subtilis WB600 by introducing the mature peptide encoding gene into the pBE2R vector with the ligation of BamH I which located at the downstream of the first amino acid of the alkaline protease mature peptide. Five insertional positions around the junction between propeptide and mature peptide were tested to detect the critical residues involved in the autoprocessing of the propeptide. Finally, it was deduced that the four critical amino acids “TTMA” which located around the 3′ terminal of the propeptide, as well as the maintenance of its integrity were necessary for the effective autoprocessing of the propeptide. Furthermore, the effect of autoprocessing of the propeptide on the formation of an active protease suggested that the propeptide was a potent inhibitor to the mature domain although it was needed in the folding of the protein before secreting.