Modification of length, hydrophobic properties and electric charge of Bacillus subtilis alpha-amylase signal peptide and their different effects on the production of secretory proteins in B. subtilis and Escherichia coli cells.
Bacillus subtilis alpha-amylase signal peptide, which consists of 33 amino acids, is functional in Escherichia coli cells. Lysine, glutamic acid, leucine, leucyl-leucine, or leucyl-leucyl-leucine was inserted between positions 28 and 29 of the alpha-amylase signal peptide using site directed mutagenesis. DNAs encoding the wild-type and modified signal peptides were then fused in-frame to DNAs encoding the mature regions of the beta-lactamase of pBR322 and a thermostable alpha-amylase. The secretion of beta-lactamase in E. coli cells was more inhibited by the modified signal peptides than that in B. subtilis cells, although the degree of inhibition varied and the inhibitory effect of each signal peptide was found to be similar in the two strains. In contrast, the difference in the inhibitory effect of each modified signal peptide was no longer detected in the case of the production of thermostable alpha-amylase, except for the insertion of glutamic acid. Nearly 50% of thermostable alpha-amylase in the precursor form was accumulated in the intracellular fraction of E. coli cells containing the DNAs for the modified signal peptides. The insertion of glutamic acid inhibited the secretion of the two enzymes in both B. subtilis and E. coli cells.
Nakamura
K
K
Institute of Biological Sciences, University of Tsukuba, Ibaraki, Japan.
Fujita
Y
Y
Itoh
Y
Y
Yamane
K
K
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000096:biosynthesis; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism*; D012329:RNA, Bacterial / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D000516:alpha-Amylases / Q000235:genetics / Q000378:metabolism*; D001618:beta-Lactamases / Q000096:biosynthesis
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D021382:Protein Sorting Signals; D012329:RNA, Bacterial; D012333:RNA, Messenger; D000516:alpha-Amylases; D001618:beta-Lactamases