Expression, purification, and characterization of the recombinant NAD-malic enzyme from Ascaris suum

The cDNA encoding the 65-kDa subunit of malic enzyme from Ascaris suum was cloned into the bacterial expression vector pKK223-3 and overproduced in Escherichia coli. A protein with a subunit molecular mass of 65,000 was expressed at a level of up to 3% of the total soluble protein in JM109, as judge...

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Bibliographic Details
Published in:Protein expression and purification 1997-06, Vol.10 (1), p.51-54
Main Authors: Chooback, L, Karsten, W E, Kulkarni, G, Nalabolu, S R, Harris, B G, Cook, P F
Format: Article
Language:English
Subjects:
Animals
Ascaris suum - enzymology
Ascaris suum - genetics
Chromatography, Liquid
Escherichia coli
Genes, Helminth
Helminth Proteins - biosynthesis
Helminth Proteins - genetics
Helminth Proteins - isolation & purification
Kinetics
Malate Dehydrogenase - biosynthesis
Malate Dehydrogenase - genetics
Malate Dehydrogenase - isolation & purification
Molecular Weight
Recombinant Fusion Proteins - biosynthesis
Recombinant Fusion Proteins - isolation & purification
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Summary:The cDNA encoding the 65-kDa subunit of malic enzyme from Ascaris suum was cloned into the bacterial expression vector pKK223-3 and overproduced in Escherichia coli. A protein with a subunit molecular mass of 65,000 was expressed at a level of up to 3% of the total soluble protein in JM109, as judged by SDS-PAGE. The enzyme was purified using column chromatography on phenyl-Sepharose followed by orange-A agarose. The purification procedure resulted in a 32-fold purification with an overall yield of 51%. The bacterially expressed enzyme exhibits kinetic constants identical to those measured for native A. suum NAD-malic enzyme.
ISSN:1046-5928
DOI:10.1006/prep.1996.0705