Cloning, expression, purification, and characterization of the 6-phosphogluconate dehydrogenase from sheep liver.
The mRNA encoding the 51-kDa subunit of 6-phosphogluconate dehydrogenase (6PGDH) from sheep liver was reverse-transcribed and amplified. The resulting cDNA was reamplified in N-terminal and C-terminal segments and spliced to generate a full-length clone, and an internal cDNA fragment was also amplified. The full-length clone containing the complete coding sequence of the 6PGDH cDNA was sequenced and found to contain two mutations and two deletions in the internal region and two mutations outside of the internal region, an A to G point mutation at position 1407 that resulted in the amino acid change Gln 445 to Arg and a silent mutation at position 1426. The internal clone was sequenced and shown to be free of any mutations; therefore the internal piece was used to replace the same region in the full-length clone to correct the mutations in this region. The mutation at position 1407 which was outside of the internal region was corrected using site-directed mutagenesis. The cDNA with the correct codon was then subcloned into the bacterial expression vector pQE-30 and overproduced in Escherichia coli strain M15. A protein with a subunit molecular weight of 51,000 was expressed at a level of about 4.5% of the total soluble protein in M15 as judged by SDS/PAGE. Cloning into pQE-30 adds six histidines and a short linker to the N-terminus of the enzyme. The recombinant 6PGDH with His-tag was purified using the Ni-NTA affinity column supplied by Qiagen. The purification procedure resulted in a homogeneous protein by SDS/PAGE with 22.4-fold purification with an overall yield of 61%. The recombinant enzyme exhibits kinetic parameters within error identical to those measured for native sheep liver enzyme.
Department of Chemistry and Biochemistry, University of Oklahoma, 620 Parrington Oval, Norman, Oklahoma, 73019, USA.
L
L
Chooback
N E
NE
Price
W E
WE
Karsten
J
J
Nelson
P
P
Sundstrom
P F
PF
Cook
D000818:Animals; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008099:Liver / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D010734:Phosphogluconate Dehydrogenase / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012756:Sheep
D011994:Recombinant Proteins; D010734:Phosphogluconate Dehydrogenase