Hum D W, MacKenzie R E
Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
Protein Eng. 1991 Apr;4(4):493-500. doi: 10.1093/protein/4.4.493.
The cDNA encoding the human trifunctional enzyme methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase was engineered to contain a prokaryotic ribosome binding site and was expressed under the bacteriophage T7 RNA polymerase promoter in Escherichia coli. Site-directed mutagenesis was used to prepare constructs that encode separately the dehydrogenase/cyclohydrolase (D/C) domain as amino acid residues 1-301, and the synthetase (Syn) domain as residues 304-935. Both domains formed active enzymes thereby demonstrating their ability to fold independently. The full-length enzyme, D/C and Syn domains were expressed at levels 4-, 55- and 3-fold higher than the specific activities found in liver. Additional mutagenesis and independent expression of domains further defined the interdomain region to include amino acids 292-310. The D/C domain was purified to homogeneity by a single affinity chromatographic step, and the full-length protein in a two-step procedure. The kinetic properties of the D/C domain appear unaltered from those of the trifunctional enzyme.
编码人三功能酶亚甲基四氢叶酸脱氢酶-亚甲基四氢叶酸环化水解酶-甲酰四氢叶酸合成酶的cDNA经改造后含有一个原核核糖体结合位点，并在噬菌体T7 RNA聚合酶启动子的控制下于大肠杆菌中表达。利用定点诱变技术构建了分别编码脱氢酶/环化水解酶（D/C）结构域（氨基酸残基1至301）和合成酶（Syn）结构域（残基304至935）的构建体。两个结构域均形成了活性酶，从而证明了它们独立折叠的能力。全长酶、D/C结构域和Syn结构域的表达水平分别比肝脏中发现的比活性高4倍、55倍和3倍。进一步的诱变和结构域的独立表达进一步确定了结构域间区域包括氨基酸292至310。D/C结构域通过一步亲和层析步骤纯化至同质，全长蛋白则通过两步法纯化。D/C结构域的动力学特性与三功能酶的动力学特性相比似乎未发生改变。
