Cloning and expression of pectin lyase gene from Erwinia carotovora in Escherichia coli.
A pectin lyase (PNL; EC 4.2.2.10) gene of Erwinia carotovora Er was cloned and expressed in Escherichia coli. The analysis of the nucleotide sequence of the 0.6 kb StuI-EcoRI fragment, which was hybridized with the mixed oligonucleotide probe for PNL gene, revealed the presence of an open reading frame (0RF) and correlated exactly with the known N-terminal 18 amino acid sequence of PNL. When a plasmid pTN2159, which has a BamHI-EcoRI fragment containing this ORF, was introduced into E. coli JM109, PNL was not expressed. When a tac-promoter was inserted in front of the ORF, PNL was efficiently expressed in E. coli. Synthesis of PNL by E. coli was also confirmed by immunoblot analysis.
Nishida
T
T
Department of Agricultural Chemistry, Faculty of Agriculture, Tohoku University, Sendai, Japan.
Suzuki
T
T
Ito
K
K
Kamio
Y
Y
Izaki
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000302:isolation & purification; D004885:Erwinia / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010957:Plasmids; D011133:Polysaccharide-Lyases / Q000235:genetics* / Q000378:metabolism
D004269:DNA, Bacterial; D011133:Polysaccharide-Lyases; C023911:pectate lyase