Sirawaraporn W, Sirawaraporn R, Cowman A F, Yuthavong Y, Santi D V
Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand.
Biochemistry. 1990 Dec 4;29(48):10779-85. doi: 10.1021/bi00500a009.
The coding sequence of the bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) from a moderately pyrimethamine-resistant strain (HB3) of Plasmodium falciparum was assembled in a pUC expression vector. The coding sequence possesses unique Nco1 and Xba1 sites which flank 243 bp of the DHFR gene that include all point mutations thus far linked to pyrimethamine resistance. Wild-type (3D7) and highly pyrimethamine-resistant (7G8) TS-DHFRs were made from this vector by cassette mutagenesis using Nco1-Xba1 fragments from the corresponding cloned TS-DHFR genes. Catalytically active recombinant TS-DHFRs were expressed in Escherichia coli, albeit at low levels. Both TS and DHFR coeluted upon gel filtration and copurified upon affinity and anion exchange chromatography. Gel filtration and SDS-PAGE indicated that the enzyme was a dimer with identical 67-kDa subunits, characteristic of protozoan TS-DHFRs. Amino-terminal sequencing gave 10 amino acids which perfectly matched the sequence predicted from the nucleotide sequence. The recombinant TS-DHFR was purified to homogeneity by 10-formylfolate affinity chromatography followed by Mono Q FPLC. The inhibition properties of pyrimethamine toward the purified recombinant enzymes show that the point mutations are the molecular basis of pyrimethamine resistance in P. falciparum.
将来自恶性疟原虫一个对乙胺嘧啶具有中等抗性的菌株（HB3）的双功能胸苷酸合成酶-二氢叶酸还原酶（TS-DHFR）的编码序列组装到一个pUC表达载体中。该编码序列具有独特的Nco1和Xba1位点，其位于DHFR基因的243 bp侧翼，其中包括迄今与乙胺嘧啶抗性相关的所有点突变。通过使用来自相应克隆的TS-DHFR基因的Nco1-Xba1片段进行盒式诱变，从该载体构建野生型（3D7）和高度乙胺嘧啶抗性（7G8）的TS-DHFR。具有催化活性的重组TS-DHFR在大肠杆菌中表达，尽管表达水平较低。TS和DHFR在凝胶过滤时共洗脱，并在亲和和阴离子交换色谱中共同纯化。凝胶过滤和SDS-PAGE表明该酶是一个由相同的67 kDa亚基组成的二聚体，这是原生动物TS-DHFR的特征。氨基末端测序得到10个氨基酸，与从核苷酸序列预测的序列完全匹配。重组TS-DHFR通过10-甲酰四氢叶酸亲和色谱，然后进行Mono Q FPLC纯化至均一。乙胺嘧啶对纯化的重组酶的抑制特性表明，这些点突变是恶性疟原虫对乙胺嘧啶抗性的分子基础。
