Expression of catalytically active radish 3-hydroxy-3-methylglutaryl coenzyme A reductase in Escherichia coli.
Two fragments of a cDNA encoding radish 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) were cloned into the vector pET-8c and expressed in Escherichia coli. The large fragment, encoding both the membrane and the cytosolic domains, was expressed at low level, essentially as an insoluble protein without enzymatic activity. In contrast, the fragment encoding only the cytosolic domain was expressed at a high level in a catalytically active form. The amount of soluble active enzyme in cell-free extracts of E. coli dramatically increased when the temperature during the induction was lowered from 37 degrees C to 22 degrees C.
Unitat de Bioquímica, Facultat de Farmacia, Universitat de Barcelona, Spain.
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Ferrer
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C
Aparicio
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Nogués
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Wettstein
T J
TJ
Bach
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Boronat
D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005822:Genetic Vectors; D006903:Hydroxymethylglutaryl CoA Reductases / Q000235:genetics* / Q000378:metabolism; D008970:Molecular Weight; D010944:Plants / Q000201:enzymology / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics; D013696:Temperature
D011994:Recombinant Proteins; D004247:DNA; D006903:Hydroxymethylglutaryl CoA Reductases