Cloning of 3-isopropylmalate dehydrogenase gene of an extreme thermophile and partial purification of the gene product.
The gene of an extreme thermophile, Thermus thermophilus HB8, which codes for a leucine biosynthetic enzyme, 3-isopropylmalate (3-IPM) dehydrogenase [EC 1.1.1.85], was cloned in Escherichia coli using pBR322 as a vector. E. coli cells carrying this recombinant plasmid, pHB2, produced the thermophilic enzyme 7-fold more than did T. thermophilus HB8 cells. When the crude extract of the pHB2-carrying cells was treated at 70 degrees C for 10 min, approximately 75% of the protein in the extract was precipitated with full activity of the thermophilic 3-IPM dehydrogenase being left in the supernatant, indicating that 4-fold purification was achieved during this process. This shows that the thermophilic 3-IPM dehydrogenase was purified 28-fold by these two procedures, cloning and heat treatment, and demonstrates that the extract from the plasmid-harboring cells is a good starting material for purification of the enzyme. Following the heat treatment, 3-IPM dehydrogenase was further purified by ammonium sulfate precipitation and DEAE-cellulose column chromatography. The enzyme preparation thus obtained contained 3-IPM dehydrogenase as a major component with a few minor impurities as shown by polyacrylamide gel electrophoresis, whereas the enzyme preparation from T. thermophilus HB8 cells obtained by the same procedures showed multiple bands on a polyacrylamide gel electrophoresis.
D050539:3-Isopropylmalate Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000235:genetics*; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D006358:Hot Temperature; D010957:Plasmids; D013824:Thermus / Q000201:enzymology / Q000235:genetics*
D000429:Alcohol Oxidoreductases; D050539:3-Isopropylmalate Dehydrogenase