M. Bigl et K. Eschrich, OVEREXPRESSION OF CATALYTICALLY ACTIVE YEAST (SACCHAROMYCES-CEREVISIAE) FRUCTOSE-1,6-BISPHOSPHATASE IN ESCHERICHIA-COLI, Biological chemistry Hoppe-Seyler, 375(3), 1994, pp. 153-160
E. coli expression plasmids for yeast (Saccharomyces cerevisiae) fruct
ose-1,6-bisphosphatase (EC 3.1.3.11) as wild-type enzyme and as lacZ f
usion protein have been constructed from a pUC vector and a fragment o
f genomic yeast DNA. Both proteins were overexpressed in E. coli strai
n TG2 as enzymatically active soluble forms and purified to homogeneit
y. While the wild-type enzyme is indistinguishable from the authentic
yeast enzyme with respect to molecular size, specific activity and kin
etic properties, the lacZ fusion protein behaves differently. Being a
tetramer like the wild-type enzyme, the specific activity of the purif
ied fusion protein is lower than that of the native enzyme. In contras
t to the wild-type enzyme the fusion fructose-1,6-bisphosphatase is no
t inhibited by excess substrate. Inhibition of the fusion protein by t
he most potent allosteric effectors of fructose-1,6-bisphosphatase, AM
P and fructose 2,6-bisphosphate, is weaker than observed with the wild
-type enzyme. The fusion protein but not the wild-type enzyme was foun
d to bind to immobilized Procion Navy H-ER. This was employed to purif
y the fusion fructose-1,6-bisphosphatase by affinity chromatography. P
olyclonal antibodies raised in rabbits against the fusion enzyme were
found to cross-react with the wild-type enzyme, but not with E. coli p
roteins. Both fructose-1,6-bisphosphatases complement the fructose-1,6
-bisphosphatase mutant DF656 of E. coli.