High-level expression of an altered cDNA encoding human isovaleryl-CoA dehydrogenase in Escherichia coli.
Isovaleryl-CoA dehydrogenase (IVD) catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA in the leucine catabolism pathway. The cDNA encoding the mature human IVD polypeptide was cloned in a prokaryotic expression vector, but the level of expression in Escherichia coli was extremely low and attempts to purify the enzyme to homogeneity were unsuccessful. To enhance expression, the nucleotide sequence of 22 codons within the 111-bp region at the 5'-end of the cDNA was altered to accommodate E. coli codon usage without altering the amino-acid coding sequence. The altered IVD cDNA was synthesized by PCR, using a primer containing the desired modifications. Following overnight induction of the E. coli transformed with this cDNA, the enzyme was purified to homogeneity using diethylaminoethyl agarose and high-pressure ceramic hydroxyapatite resins. IVD activity was increased 165-fold in the crude extract of cells containing the modified cDNA, as compared to that containing the wild-type cDNA.
Mohsen
A W
AW
Department of Medical Genetics, Mayo Clinic, Rochester, MN 55905, USA.
Vockley
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D003062:Codon / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D017886:Durapatite; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D015870:Gene Expression; D005796:Genes; D006801:Humans; D050769:Isovaleryl-CoA Dehydrogenase; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D044925:Oxidoreductases Acting on CH-CH Group Donors; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D012685:Sepharose; D013056:Spectrophotometry, Ultraviolet
D003062:Codon; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D005182:Flavin-Adenine Dinucleotide; D012685:Sepharose; D017886:Durapatite; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; D050769:Isovaleryl-CoA Dehydrogenase