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Biotechnology and Applied Biochemistry (2003) 37, (187–194) (Printed in Great Britain)

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Overexpression of the pectin lyase gene of Pseudomonas marginalis in Escherichia coli and purification of the active enzyme

Rigini Papi and Dimitrios Kyriakidis¹

Laboratory of Biochemistry, Department of Chemistry, Aristotle University of Thessaloniki, Thessaloniki, 54006, Greece

Key words: affinity chromatography, hexahistidine-tagged protein, plasmid DNA retardation, pectin lyase inhibitor.

Abbreviations used: PNL, pectin lyase; Ni²⁺-NTA, Ni²⁺-nitrilotriacetate; IPTG, isopropyl b-D-thiogalactoside.

¹To whom correspondence should be addressed (e-mail kyr@chem.auth.gr).

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 Ni²⁺-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.

Received 12 June 2002/7 October 2002; accepted 8 November 2002

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