High-level expression of porcine fructose-1,6-bisphosphatase in Escherichia coli: purification and characterization of the enzyme.

A cDNA encoding porcine fructose-1,6-bisphosphatase was isolated from total pig liver RNA. The enzyme's coding sequence was fused to the bacteriophage T7 gene 10 promoter and transcription terminator sequence and expressed in E. coli under control of the T7 RNA polymerase. Induced cells contain elevated levels of fructose-1,6-bisphosphatase enzymatic activity and an abundant 37,000 dalton protein. The enzyme was purified to apparent homogeneity and judged identical to wild-type porcine fructose-1,6-bisphosphatase. The kinetic parameters are similar to those reported for the pig kidney enzyme. The N-terminal amino acid sequence is identical to the predicted sequence and the kinetic parameters are consistent with freedom from proteolysis. As estimated from enzymatic activity and visual inspection of coomassie blue-stained SDS-PAGE gels, porcine fructose-1,6-bisphosphatase constitutes as much as 20% of the soluble protein from the over-expressing E. coli strain.