Mandrup S, Højrup P, Kristiansen K, Knudsen J
Institute of Biochemistry, Odense University, Denmark.
Biochem J. 1991 Jun 15;276 ( Pt 3)(Pt 3):817-23. doi: 10.1042/bj2760817.
A synthetic gene encoding the 86 amino acid residues of mature acyl-CoA-binding protein (ACBP), and the initiating methionine was constructed. The synthetic gene was assembled from eight partially overlapping oligonucleotides. Codon usage and nucleotides surrounding the ATG translation-initiation codon were chosen to allow efficient expression in Escherichia coli as well as in yeast. The synthetic gene was inserted into the expression vector pKK223-3 and expressed in E. coli. In maximally induced cultures, recombinant ACBP constitutes 12-15% of total cellular protein. A fraction highly enriched for recombinant ACBP was obtained by extracting induced E. coli cells with 1 M-acetic acid. Recombinant ACBP was purified to homogeneity by successive use of gel-filtration chromatography, ion-exchange chromatography and reverse-phase h.p.l.c. Recombinant ACBP differed from native ACBP by lacking the N-terminal acetyl group. The acyl-CoA-binding characteristics of recombinant ACBP did not differ from those of native ACBP, and the two proteins showed the same ability to induce medium-chain acyl-CoA synthesis by goat mammary-gland fatty acid synthetase. It was concluded that the N-terminal acetyl group is not important for acyl-CoA binding.
构建了一个编码成熟酰基辅酶A结合蛋白（ACBP）86个氨基酸残基以及起始甲硫氨酸的合成基因。该合成基因由八个部分重叠的寡核苷酸组装而成。选择密码子使用情况以及ATG翻译起始密码子周围的核苷酸，以使其在大肠杆菌和酵母中均能高效表达。将合成基因插入表达载体pKK223-3并在大肠杆菌中表达。在诱导程度最高的培养物中，重组ACBP占总细胞蛋白的12% - 15%。通过用1M乙酸提取诱导的大肠杆菌细胞，获得了高度富集重组ACBP的部分。通过连续使用凝胶过滤色谱、离子交换色谱和反相高效液相色谱，将重组ACBP纯化至同质。重组ACBP与天然ACBP的不同之处在于缺少N端乙酰基。重组ACBP的酰基辅酶A结合特性与天然ACBP无异，并且这两种蛋白在诱导山羊乳腺脂肪酸合成酶合成中链酰基辅酶A方面表现出相同的能力。得出的结论是，N端乙酰基对于酰基辅酶A结合并不重要。
