CLONING, EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF THE 6-PHOSPHOGLUCONATE DEHYDROGENASE FROM SHEEP LIVER

The mRNA encoding the 51-kDa subunit of 6-phosphogluconate dehydrogena se (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 frag ment 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 mutatio ns outside of the internal region, an A to G point mutation at positio n 1407 that resulted in the amino acid change Gln 445 to Arg and a sil ent mutation at position 1426, The internal clone was sequenced and sh own 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 mu tations in this region. The mutation at position 1407 which was outsid e of the internal region was corrected using site-directed mutagenesis . The cDNA with the correct codon was then subcloned into the bacteria l expression vector pQE-30 and overproduced in Escherichia coli strain M15. A protein with a subunit molecular weight of 51,000 was expresse d at a level of about 4.5% of the total soluble protein in M15 as judg ed by SDS/PAGE. Cloning into pQE-30 adds six histidines and a short li nker to the N-terminus of the enzyme. The recombinant 6PGDH with His-t ag was purified using the Ni-NTA affinity column supplied by Qiagen, T he purification procedure resulted in a homogeneous protein by SDS/PAG E with 22.4-fold purification with an overall yield of 61%, The recomb inant enzyme exhibits kinetic parameters within error identical to tho se measured for native sheep liver enzyme. (C) 1998 Academic Press.