OVEREXPRESSION OF CATALYTICALLY ACTIVE YEAST (SACCHAROMYCES-CEREVISIAE) FRUCTOSE-1,6-BISPHOSPHATASE IN ESCHERICHIA-COLI

Authors
Citation
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
Citations number
34
Categorie Soggetti
Biology
ISSN journal
01773593
Volume
375
Issue
3
Year of publication
1994
Pages
153 - 160
Database
ISI
SICI code
0177-3593(1994)375:3<153:OOCAY(>2.0.ZU;2-Z
Abstract
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.