Cloning and high-level expression of chitinase-encoding gene of Streptomyces plicatus.
A chitinase (Cht)-encoding gene from Streptomyces plicatus was previously cloned and expressed in Escherichia coli [Robbins et al., J. Biol. Chem., 263 (1988) 442-447]. We have sequenced this gene, compared its sequence with other genes encoding Cht and have explored its expression and regulation when reintroduced into Streptomyces lividans on multicopy plasmids. We have also cloned two other Streptomyces Cht-encoding genes and a beta-hexosaminidase-encoding gene in E. coli by expression in the lambda ZAP-Bluescript vector. The hexosaminidase and one of the Chts were expressed directly from the genomic library in E. coli at a high level as chimeric fusions with the beta-galactosidase alpha-complementing peptide encoded by the vector. Direct cloning and high-level expression of such chimeric proteins, which overcomes the difficulties associated with expressing Streptomyces genes in E. coli, should generally be possible wherever large numbers of transformants can be conveniently screened.
Robbins
P W
PW
Center for Cancer Research, M.I.T., Cambridge 02139.
Overbye
K
K
Albright
C
C
Benfield
B
B
Pero
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002688:Chitinases / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics*; D001619:beta-N-Acetylhexosaminidases / Q000235:genetics / Q000378:metabolism
D004269:DNA, Bacterial; D002688:Chitinases; D001619:beta-N-Acetylhexosaminidases