Fan Y X, Wang B, Jing G Z
National Laboratory of Biomacromolecules, Academia Sinica, Beijing, China.
Protein Expr Purif. 1997 Mar;9(2):246-52. doi: 10.1006/prep.1996.0676.
Chinese hamster dihydrofolate reductase (ch-DHFR) was overexpressed in Escherichia coli DH5 alpha under the transcriptional control of PRPL promoters regulated by temperature-sensitive repressors. The desired recombinant product is soluble and constitutes about 30% of the total soluble proteins of the bacterial cell. With repeated cycles of freezing and thawing as a first step, the purification of the recombinant ch-DHFR to homogeneity requires only one further step, gel filtration on a Sephadex G-75 column with 85-90% enzyme recovery, two to three times higher than that obtained with the commonly used affinity chromatography on a methotrexate-Sepharose column. The purified enzyme migrates as a single protein band on SDS-polyacrylamide gel electrophoresis with approximate mass of 23 kDa, in accord with that calculated from the DNA sequence. The initiation methionine residue at the N-terminus of the enzyme is completely removed by E. coli methionine aminopeptidase as judged by amino-terminal analysis. The steady-state kinetic parameters, dissociation constants for binary complexes of dihydrofolate, NADPH, and methotrexate with ch-DHFR, and the inhibitor constant of methotrexate have also been determined. The enzyme is activated about 4-fold in 3 M urea and about 2.5-fold in 0.5 M guanidine hydrochloride.
中国仓鼠二氢叶酸还原酶（ch-DHFR）在温度敏感型阻遏物调控的PRPL启动子转录控制下，于大肠杆菌DH5α中过表达。所需的重组产物是可溶的，约占细菌细胞总可溶性蛋白的30%。以反复冻融作为第一步，将重组ch-DHFR纯化至均一性仅需再进行一步，即在Sephadex G-75柱上进行凝胶过滤，酶回收率为85 - 90%，比在甲氨蝶呤-琼脂糖柱上常用的亲和层析法获得的回收率高两到三倍。纯化后的酶在SDS-聚丙烯酰胺凝胶电泳上迁移为单一蛋白条带，其近似质量为23 kDa，与根据DNA序列计算的结果一致。通过氨基末端分析判断，该酶N端的起始甲硫氨酸残基被大肠杆菌甲硫氨酸氨肽酶完全去除。还测定了稳态动力学参数、二氢叶酸、NADPH和甲氨蝶呤与ch-DHFR二元复合物的解离常数以及甲氨蝶呤的抑制常数。该酶在3 M尿素中活性约提高4倍，在0.5 M盐酸胍中活性约提高2.5倍。
