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Over-expression, purification, and characterization of recombinant NAD-malic enzyme from Escherichia coli K12
- Source :
-
Protein Expression & Purification . May2007, Vol. 53 Issue 1, p97-103. 7p. - Publication Year :
- 2007
-
Abstract
- Abstract: NAD+-dependent malic enzyme (NAD-ME) gene from Escherichia coli K12 was inserted into an expression vector pET24b(+) and transformed into E. coli BL21 (DE3). Recombinant NAD-ME was expressed upon IPTG induction, purified with affinity chromatography, and biochemically characterized. The results showed that recombinant NAD-ME could be produced mainly in a soluble form. The monomeric molecular weight of recombinant NAD-ME was about 65kDa, whereas monomer, homotetramer, and homooctamer were formed in solution as revealed by nondenaturing polyacrylamide gel electrophoresis analysis. Finally, the K m values of NAD-ME for l-malate and NAD were determined as 0.420±0.174 and 0.097±0.038mM, respectively, at pH 7.2. By using this over-expression and purification system, recombinant E. coli K12 NAD-ME can now be obtained in large quantity necessary for further biochemical characterization and applications. [Copyright &y& Elsevier]
- Subjects :
- *GENE expression
*ESCHERICHIA coli
*ENZYME kinetics
*RECOMBINANT viruses
Subjects
Details
- Language :
- English
- ISSN :
- 10465928
- Volume :
- 53
- Issue :
- 1
- Database :
- Academic Search Index
- Journal :
- Protein Expression & Purification
- Publication Type :
- Academic Journal
- Accession number :
- 24148608
- Full Text :
- https://doi.org/10.1016/j.pep.2006.11.017