Chin M S, Wang C C
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143.
Mol Biochem Parasitol. 1994 Feb;63(2):221-9. doi: 10.1016/0166-6851(94)90058-2.
We have cloned and expressed the full-length gene encoding the hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase) from the anaerobic protozoan parasite Tritrichomonas foetus. This enzyme is essential in nucleic acid metabolism of T. foetus because the parasite is unable to synthesize purine nucleotides de novo and relies on the HGXPRTase activities for its purine requirements. Initially, a cDNA clone encoding part of the HGXPRTase was isolated by complementation of an Escherichia coli mutant, SO609, with a cDNA library of T. foetus. Northern blot analysis identified a single mRNA band of approximately 700-800 bases. The full-length genomic clone was then isolated and identified to have an open reading frame of 549 bp encoding an 183-amino acid sequence with an estimated size of 21.1 kDa. The sequence is only 27.3% identical to that of the human HGPRTase. The T. foetus HGXPRTase gene was subsequently cloned into the pBAce vector for expression in E. coli. This construct yields completely soluble and enzymatically active recombinant T. foetus HGXPRTase, which constitutes approximately 20% of the total cellular protein of the transformed E. coli. It has the same molecular weight as the authentic native enzyme, and the N-terminal amino acid sequence of the recombinant enzyme is identical to that predicted from the open reading frame. The high expression of this apparently native T. foetus HGXPRTase will provide large quantities of purified protein, necessary for detailed kinetic and structural analysis of this enzyme for its potential value as a target for antitrichomonial chemotherapy. To our knowledge, this is also the first time a gene from T. foetus was cloned and expressed.
我们已经克隆并表达了编码来自厌氧原生动物寄生虫胎儿三毛滴虫次黄嘌呤 - 鸟嘌呤 - 黄嘌呤磷酸核糖转移酶（HGXPRTase）的全长基因。这种酶在胎儿三毛滴虫的核酸代谢中至关重要，因为该寄生虫无法从头合成嘌呤核苷酸，其嘌呤需求依赖于HGXPRTase的活性。最初，通过用胎儿三毛滴虫的cDNA文库互补大肠杆菌突变体SO609，分离出编码部分HGXPRTase的cDNA克隆。Northern印迹分析鉴定出一条约700 - 800个碱基的单一mRNA条带。然后分离出全长基因组克隆，鉴定其具有549 bp的开放阅读框，编码一个183个氨基酸的序列，估计大小为21.1 kDa。该序列与人HGPRTase的序列仅有27.3%的同一性。随后将胎儿三毛滴虫HGXPRTase基因克隆到pBAce载体中，以便在大肠杆菌中表达。该构建体产生完全可溶且具有酶活性的重组胎儿三毛滴虫HGXPRTase，其占转化大肠杆菌总细胞蛋白的约20%。它与天然酶具有相同的分子量，并且重组酶的N端氨基酸序列与从开放阅读框预测的序列相同。这种明显天然的胎儿三毛滴虫HGXPRTase的高表达将提供大量纯化蛋白，这对于详细分析该酶的动力学和结构以确定其作为抗滴虫化疗靶点的潜在价值是必要的。据我们所知，这也是首次克隆并表达来自胎儿三毛滴虫的基因。
