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Overexpression of the pectin lyase gene of Pseudomonas marginalis in Escherichia coli and purification of the active enzyme
Author(s) -
Papi Rigini,
Kyriakidis Dimitrios
Publication year - 2003
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1042/ba20020055
Subject(s) - escherichia coli , biochemistry , biology , microbiology and biotechnology , enzyme , plasmid , dna , molecular mass , agarose , recombinant dna , pectin lyase , gene , pectinase
A pectin lyase gene ( pnl ) of Pseudomonas marginalis was cloned and overexpressed in Escherichia coli BL21(DE3). The pnl gene was amplified by PCR, inserted into pET29c with a six‐His tag and the overproduced active enzyme was purified almost to homogeneity using a Ni 2+ ‐nitrilotriacetate–agarose column. The purified pectin lyase (PNL; EC 4.2.2.10, family 1) is inhibited by NAD + (at concentrations above 0.25 mM), NADH or dithiothreitol. Evidence for the existence of a heat‐labile protein inhibitor of PNL is also reported. The DNA‐binding ability of PNL was demonstrated by DNA‐retardation experiments. The partially purified enzyme was incubated with plasmid DNA and the complex was shifted to a higher molecular mass. Analysis of the electroeluted proteins from the protein–DNA complex revealed that one of the electroeluted protein bands was PNL. Antibodies against the overexpressed PNL were also prepared and partially purified.