[Cloning expression and purification of glycerol dehydrogenase from Klebsiella pneumoniae].
The gldA gene coding glycerol dehydrogenase (GDH) was amplified by PCR with the genomic DNA of Klebsiella pneumoniae as the template. The gldA were inserted in pMD-18T to construct the recombinant cloning vector pMD-gldA. After the DNA sequence was determined, the gldA was subcloned into expression vector pET-32a (+) to construct the recombinant expression vector pET-32gldA. Upon lactose induction, soluble GDH was over-produced by E. coli BL21 (DE3) harboring the expression construct. Recombinant GDH purified by Ni-NTA affinity chromatography showed a single band about 54 kD on SDS-PAGE gel, and the specified activity was about 188 u/mg, the purification fold is 3 times and the activity recovery is 67.5%.
Zhang
Tingting
T
Key Laboratory of Industrial Biotechnology Fujian Province, Huaqiao University, Quanzhou 362021, China.
Fang
Baishan
B
Wang
Geng
G
Wang
Feifei
F
D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007711:Klebsiella pneumoniae / Q000201:enzymology* / Q000235:genetics*; D013401:Sugar Alcohol Dehydrogenases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D004269:DNA, Bacterial; D013401:Sugar Alcohol Dehydrogenases; C020672:glycerol dehydrogenase