Overexpression and purification of fructose-1-phosphate kinase from Escherichia coli: application to the assay of fructose 1-phosphate.
Fructose 1-phosphate is a metabolite that plays a regulatory role in liver and is best measured using an assay based on its conversion to fructose 1,6-bisphosphate by a bacterial fructose-1-phosphate kinase (Fru1PK). The open reading frame encoding Escherichia coli Fru1PK has been introduced in an expression plasmid (pET3a) based on the T7 promoter-driven system, which was used to overexpress the enzyme. The conditions for the production of soluble Fru1PK were optimized. The purification procedure used involved ammonium sulfate precipitation and chromatography on DEAE-Sepharose and was aimed mostly at stabilizing the enzyme and at freeing Fru1PK from bacterial contaminants that could interfere in the fructose 1-phosphate assay. From a 1-liter culture, more than 50 mg protein is obtained. This preparation can be used in an enzymatic assay that measures specifically fructose 1-phosphate in tissue extracts.
Veiga-da-Cunha
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M
Laboratory of Physiological Chemistry, ICP and Université Catholique de Louvain, Brussels, B-1200, Belgium.
Hoyoux
A
A
Van Schaftingen
E
E
Houyoux
A
A
D000818:Animals; D002847:Chromatography, Agarose; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D005631:Fructokinases / Q000378:metabolism; D006597:Fructose-Bisphosphatase / Q000378:metabolism; D005634:Fructose-Bisphosphate Aldolase / Q000378:metabolism; D005636:Fructosephosphates / Q000378:metabolism*; D016366:Open Reading Frames; D010732:Phosphofructokinase-1 / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids; D051381:Rats; D013053:Spectrophotometry; D014020:Tissue Extracts
D005636:Fructosephosphates; D014020:Tissue Extracts; C032284:fructose-1-phosphate; D005631:Fructokinases; D010732:Phosphofructokinase-1; C046173:fructokinase; C068652:1-phosphofructokinase; D006597:Fructose-Bisphosphatase; D005634:Fructose-Bisphosphate Aldolase