Kireeva M L, Murzina N V, Murzin A G, Uversky V N, Gryaznova O I, Gudkov A T
Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region.
Protein Eng. 1994 Nov;7(11):1373-7. doi: 10.1093/protein/7.11.1373.
Three circularly permuted variants of Escherichia coli dihydrofolate reductase genes were constructed. Linkers coding tri- and pentapeptides were used to connect the natural 5'- and 3'-terminal ends. Only one variant of circularly permuted protein with tripeptide linker and the cleavage of the peptide bond between 107 and 108 amino acid residues was produced in a good yield. The expressed protein was insoluble in the cells, but at pH 8.0 and higher the isolated protein was soluble. Enzymatic assay and physical studies have shown that permuted dihydrofolate reductase has a destabilized tertiary structure. Only the addition of the natural substrates or inhibitors lead to the protein with the native-like structure and functional activity.
构建了大肠杆菌二氢叶酸还原酶基因的三种环状排列变体。编码三肽和五肽的接头用于连接天然的5'和3'末端。仅产生了一种带有三肽接头且在107和108个氨基酸残基之间的肽键被切割的环状排列蛋白变体，产量良好。表达的蛋白在细胞中不溶，但在pH 8.0及更高时，分离出的蛋白可溶。酶活性测定和物理研究表明，环状排列的二氢叶酸还原酶具有不稳定的三级结构。只有添加天然底物或抑制剂才能使蛋白具有类似天然的结构和功能活性。
