Secretion of active subtilisin YaB by a simultaneous expression of separate pre-pro and pre-mature polypeptides in Bacillus subtilis.
Alkaline elastase YaB, produced by alkalophilic Bacillus YaB, is an extracellular serine protease having 55% homology to subtilisin BPN' and thus could be called subtilisin YaB. It is synthesized as a 378-amino acid preproenzyme and secreted into the culture medium as a 265-amino acid mature protease. To examine if the pro-peptide of subtilisin YaB functions in trans to guide the folding of secreted subtilisin YaB in vivo, we made genes encoding the pre-pro, pro and pre-mature portions and placed them under the control of the spac-1 promoter on a multi-copy plasmid. When simultaneous expression in Bacillus subtilis of both the pre-pro and pre-mature genes was induced with 0.5 mM isopropyl-1-thio-beta-D-galactopyranoside (IPTG), protease activity was detected in the medium. On the other hand, we could not detect protease activity when the expression of either the pre-mature gene alone or both the pro and pre-mature genes was induced. From these results, we concluded that the pro region functions in trans and outside the cells for the proper folding and activation of the enzyme.
Chang
Y C
YC
Department of Biotechnology, University of Tokyo, Yayoi, Bunkyo-ku, Japan.
Kadokura
H
H
Yoda
K
K
Yamasaki
M
M
D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004792:Enzyme Precursors / Q000096:biosynthesis* / Q000378:metabolism; D004926:Escherichia coli; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D013381:Subtilisins / Q000096:biosynthesis* / Q000378:metabolism
D017931:DNA Primers; D004792:Enzyme Precursors; D011994:Recombinant Proteins; D013381:Subtilisins; C060795:alkaline elastase YaB