Mohsen A W, Vockley J
Department of Medical Genetics, Mayo Clinic, Rochester, MN 55905, USA.
Gene. 1995 Jul 28;160(2):263-7. doi: 10.1016/0378-1119(95)00256-6.
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.
异戊酰辅酶A脱氢酶（IVD）在亮氨酸分解代谢途径中催化异戊酰辅酶A转化为3-甲基巴豆酰辅酶A。编码成熟人IVD多肽的cDNA被克隆到原核表达载体中，但在大肠杆菌中的表达水平极低，并且将该酶纯化至均一性的尝试未成功。为了提高表达，改变了cDNA 5'端111bp区域内22个密码子的核苷酸序列，以适应大肠杆菌的密码子使用情况，同时不改变氨基酸编码序列。使用含有所需修饰的引物通过PCR合成了改变后的IVD cDNA。用该cDNA转化大肠杆菌过夜诱导后，使用二乙氨基乙基琼脂糖和高压陶瓷羟基磷灰石树脂将该酶纯化至均一性。与含有野生型cDNA的细胞粗提物相比，含有修饰后cDNA的细胞粗提物中IVD活性增加了165倍。
