Eakin A E, Bouvier J, Sakanari J A, Craik C S, McKerrow J H
Department of Pathology, University of California, San Francisco.
Mol Biochem Parasitol. 1990 Feb;39(1):1-8. doi: 10.1016/0166-6851(90)90002-4.
Cysteine protease gene fragments from three protozoan parasites Trypanosoma cruzi, Trypanosoma brucei, and Entamoeba histolytica were amplified by the polymerase chain reaction (PCR) from genomic DNA using degenerate oligonucleotide primers. The primers used for the amplification were designed based upon amino acid sequences flanking the active site cysteine and asparagine residues that are conserved in the eukaryotic cysteine proteases analyzed to date. The amplified DNA fragments, representing approximately 70% of the coding regions of the cysteine protease genes, were subcloned and sequenced. Sequence analysis and alignment showed significant sequence similarity to other members of the eukaryotic cysteine protease family (45% identical to chicken cathepsin L) and conservation of the cysteine, histidine, and asparagine residues which form the catalytic triad. These gene fragments provide molecular probes for further analysis of the structure and function of these important metabolic enzymes.
使用简并寡核苷酸引物通过聚合酶链反应（PCR）从基因组DNA中扩增来自三种原生动物寄生虫克氏锥虫、布氏锥虫和溶组织内阿米巴的半胱氨酸蛋白酶基因片段。用于扩增的引物是根据迄今为止所分析的真核生物半胱氨酸蛋白酶中活性位点半胱氨酸和天冬酰胺残基侧翼的氨基酸序列设计的。代表半胱氨酸蛋白酶基因编码区约70%的扩增DNA片段被亚克隆并测序。序列分析和比对显示与真核生物半胱氨酸蛋白酶家族的其他成员具有显著的序列相似性（与鸡组织蛋白酶L有45%的同一性），并且形成催化三联体的半胱氨酸、组氨酸和天冬酰胺残基具有保守性。这些基因片段为进一步分析这些重要代谢酶的结构和功能提供了分子探针。
