Over-expression, purification, and characterization of recombinant NAD-malic enzyme from Escherichia coli K12.
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 65 kDa, 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+/-0.038 mM, 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.
Wang
Jinxia
J
Dalian Institute of Chemical Physics, CAS, Dalian 116023, China.
Tan
Haidong
H
Zhao
Zongbao Kent
ZK
D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D048168:Escherichia coli K12 / Q000201:enzymology*; D015870:Gene Expression; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D007544:Isopropyl Thiogalactoside / Q000096:biosynthesis; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000235:genetics* / Q000378:metabolism*; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D014170:Transformation, Genetic
D011994:Recombinant Proteins; D009243:NAD; D007544:Isopropyl Thiogalactoside; D008291:Malate Dehydrogenase; C046446:malate dehydrogenase-(oxaloacetate-decarboxylating) (NAD+)