Hsu T Y, Liu M W, Chang Y R, Pai C Y, Liu M Y, Yang C S, Chen J Y
Graduate Institute of Microbiology, College of Medicine, National Taiwan University, Taipei, Taiwan.
J Gen Virol. 1996 Aug;77 ( Pt 8):1893-9. doi: 10.1099/0022-1317-77-8-1893.
Thymidine kinase (TK) activity was detected following expression of the TK gene of Epstein-Barr virus (EBV) using the pET expression plasmid and E. coli BL21 (DE3)pLysS. To study the amino acid residues required at the C terminus of the EBV TK protein for enzymatic activity, a series of C-terminal deletion mutants was generated by direct truncation, linker insertion or PCR mutagenesis to create stop codons at particular sites. Deletion of nine residues from the C terminus caused a 35% reduction in TK activity, while a ten-residue deletion completely abolished the activity. A single point mutation at residue Cys570, corresponding to Cys336 of herpes simplex virus TK, did not alter the TK activity. Single amino acid changes within the last seven to ten residues also did not affect activity. The results indicate that maintenance of the conformation of the C terminus is important for enzyme activity.
使用pET表达质粒和大肠杆菌BL21(DE3)pLysS，在表达爱泼斯坦-巴尔病毒(EBV)的胸苷激酶(TK)基因后检测到TK活性。为了研究EBV TK蛋白C末端对于酶活性所需的氨基酸残基，通过直接截短、接头插入或PCR诱变产生了一系列C末端缺失突变体，以在特定位点产生终止密码子。从C末端缺失九个残基导致TK活性降低35%，而缺失十个残基则完全消除了活性。对应于单纯疱疹病毒TK的Cys336的Cys570残基处的单点突变没有改变TK活性。最后七至十个残基内的单个氨基酸变化也不影响活性。结果表明，C末端构象的维持对酶活性很重要。
