Cloning and expression of pectin lyase gene from Erwinia carotovora in Escherichia coli |
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| Authors: | T Nishida T Suzuki K Ito Y Kamio K Izaki |
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| Affiliation: | Department of Agricultural Chemistry, Faculty of Agriculture, Tohoku University, Sendai, Japan. |
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| Abstract: | 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. |
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