Cloning and expression in Escherichia coli of the gene encoding the Proteus vulgaris chondroitin ABC lyase.
The structural gene encoding chondroitin ABC lyase from Proteus vulgaris was cloned and sequenced. This gene consists of a single open reading frame of 3,063 bp, including a sequence (72 bp) for a possible secretory protein leader peptide, preceded by a Shine-Dalgarno ribosomal binding site. Promoter-like and rho-independent terminator sequences were detected upstream and downstream of the open reading frame, respectively. The G+C content of the coding region was 38.6%. The transcription startpoint was located 41-bp upstream of the initiation codon (ATG). Chondroitin ABC lyase is composed of 997 amino acids, and has a relative molecular mass of 112,635. When the 5.2-kb fragment containing the 1.2-kb upstream from the gene was inserted into pSTV29, and cloned in Escherichia coli, chondroitin ABC lyase was induced in the medium containing chondroitin-6-sulfate as the carbon source. On the other hand, when a 4.2-kb fragment containing only 0.2 kb upstream was inserted into pSTV29(pCHS delta 6), and pCHS delta 6 was introduced into E. coli, the enzyme was constitutively produced, even in medium containing glucose as the carbon source. By immunoblot analysis, the polypeptide synthesized by E. coli cells carrying pCHS delta 6 appeared to be the same as that of the purified chondroitin ABC lyase from P. vulgaris.
Sato
N
N
Central Research Institute, Maruha Corporation, Ibaraki, Japan.
Shimada
M
M
Nakajima
H
H
Oda
H
H
Kimura
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D002808:Chondroitin Lyases / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000302:isolation & purification; D004790:Enzyme Induction; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics*; D015698:Genomic Library; D008969:Molecular Sequence Data; D011514:Proteus vulgaris / Q000201:enzymology* / Q000235:genetics; D012329:RNA, Bacterial / Q000032:analysis; D015183:Restriction Mapping; D017421:Sequence Analysis; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D012329:RNA, Bacterial; D002808:Chondroitin Lyases