Kolm R H, Stenberg G, Widersten M, Mannervik B
Department of Biochemistry, Uppsala University, Sweden.
Protein Expr Purif. 1995 Jun;6(3):265-71. doi: 10.1006/prep.1995.1034.
A cDNA clone, lambda GTHP1del, encoding glutathione transferase (GST) P1-1, was isolated from a human K562 erythroleukemia cell line cDNA library. The coding sequence was lacking the codons for the N-terminal 34 amino acids. A DNA segment was designed in order to obtain the missing portion and a structure representing the entire protein. The synthetic DNA sequence was constructed to achieve efficient base pairing with Escherichia coli 16S ribosomal RNA, avoidance of internal secondary structure, and optimal codon usage for high-level protein expression in accord with the known preferences in E. coli. The truncated GST P1-1 cDNA sequence and the synthetic segment were ligated into a plasmid to give an inducible expression system. Among the resulting clones a limited number was selected by immunodetection for highest yield of GST P1-1. Maximal expression was obtained from a spontaneously mutated sequence with altered as well as deleted bases as compared to the original construct. This clone, pKXHP1, allowed heterologous expression in E. coli in yields of > 200 mg enzyme per liter culture medium. The physicochemical and catalytic properties of the recombinant protein were indistinguishable from those of the enzyme purified from human placenta.
从人K562红白血病细胞系cDNA文库中分离出一个编码谷胱甘肽S-转移酶（GST）P1-1的cDNA克隆，λGTHP1del。其编码序列缺少N端34个氨基酸的密码子。设计了一个DNA片段以获得缺失部分并构建代表完整蛋白质的结构。合成DNA序列的构建旨在实现与大肠杆菌16S核糖体RNA的有效碱基配对，避免内部二级结构，并根据大肠杆菌中已知的偏好使用最佳密码子以实现高水平的蛋白质表达。将截短的GST P1-1 cDNA序列与合成片段连接到质粒中，得到一个可诱导表达系统。通过免疫检测从所得克隆中选择了有限数量的克隆，以获得最高产量的GST P1-1。与原始构建体相比，从具有改变和缺失碱基的自发突变序列中获得了最大表达。该克隆pKXHP1允许在大肠杆菌中进行异源表达，每升培养基中酶的产量>200mg。重组蛋白的物理化学和催化特性与从人胎盘中纯化的酶的特性无法区分。
