High expression in Escherichia coli of the gene coding for dihydrofolate reductase of the extremely halophilic archaebacterium Haloferax volcanii. Reconstitution of the active enzyme and mutation studies.
The gene coding for the enzyme dihydrofolate reductase of the extremely halophilic archaebacterium Haloferax volcanii was recombined into the Escherichia coli expression vector pET11d. Following induction, the enzyme was produced in large quantities and accumulated in the cells in an insoluble form. The enzymic activity could be efficiently reconstituted by dissolving the aggregate in 6 M guanidine hydrochloride followed by dilution into salt solutions. Mutants were produced in which Lys30 was converted to Leu (K30L), Lys31 was converted to Ala (K31A) and a double mutant in which both lysines were converted (K30L, K31A). The mutated enzymes were produced in E. coli, activated and purified to homogeneity. The effect of the salt concentration on the steady-state kinetic parameters was determined. It was found that the salt concentration affects the Km but not kcat of the various mutants.
Blecher
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O
Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Israel.
Goldman
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S
Mevarech
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M
D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D005822:Genetic Vectors; D006216:Halobacteriaceae / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D007930:Leucine / Q000737:chemistry; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D011994:Recombinant Proteins; D013762:Tetrahydrofolate Dehydrogenase; D007930:Leucine; D008239:Lysine