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.
Department of Agricultural Chemistry, Faculty of Agriculture, Tohoku University, Sendai, Japan.
T
T
Nishida
T
T
Suzuki
K
K
Ito
Y
Y
Kamio
K
K
Izaki
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