Rosenthal P J, Nelson R G
Department of Medicine, San Francisco General Hospital, University of California.
Mol Biochem Parasitol. 1992 Mar;51(1):143-52. doi: 10.1016/0166-6851(92)90209-3.
We have previously identified a 28-kDa cysteine proteinase of Plasmodium falciparum trophozoites that appears to be an essential malarial hemoglobinase and a potential target for antimalarial chemotherapy. The trophozoite cysteine proteinase (TCP) shares a number of biochemical properties with the lysosomal cysteine proteinase cathepsin L. To isolate the gene encoding TCP, we synthesized degenerate oligonucleotides based on two amino acid sequences of cathepsin L that are well conserved among papain-family cysteine proteinases, and used the oligonucleotides to prime the polymerase chain reaction (PCR) with P. falciparum genomic DNA. A 549-bp DNA fragment was amplified by PCR. This fragment was used as a hybridization probe to screen a lambda gt11 library of P. falciparum genomic DNA and isolate a 1.8-kb genomic clone (C1.8) that encoded an intact malarial cysteine proteinase gene. The sequence of C1.8 predicted a 67-kDa protein containing a typical signal sequence, a large pro sequence, and a 26.8-kDa mature proteinase with 37% amino acid identity to cathepsin L. Antisera directed against a peptide encoded by C1.8 recognized a 28-kDa trophozoite protein on immunoblots. In a Northern analysis, C1.8 hybridized predominantly with RNA from rings, the life-cycle stage immediately preceding the trophozoite stage. Taken together, these results strongly suggest that the P. falciparum cysteine proteinase gene we have isolated and characterized encodes TCP.
我们先前已鉴定出恶性疟原虫滋养体的一种28 kDa半胱氨酸蛋白酶，它似乎是一种必需的疟疾血红蛋白酶，也是抗疟化疗的潜在靶点。滋养体半胱氨酸蛋白酶（TCP）与溶酶体半胱氨酸蛋白酶组织蛋白酶L具有许多生化特性。为了分离编码TCP的基因，我们根据组织蛋白酶L的两个氨基酸序列合成了简并寡核苷酸，这两个序列在木瓜蛋白酶家族半胱氨酸蛋白酶中高度保守，并使用这些寡核苷酸以恶性疟原虫基因组DNA为模板进行聚合酶链反应（PCR）。通过PCR扩增出一个549 bp的DNA片段。该片段用作杂交探针，筛选恶性疟原虫基因组DNA的λgt11文库，并分离出一个1.8 kb的基因组克隆（C1.8），该克隆编码一个完整的疟疾半胱氨酸蛋白酶基因。C1.8的序列预测编码一个67 kDa的蛋白质，包含一个典型的信号序列、一个大的前体序列和一个26.8 kDa的成熟蛋白酶，与组织蛋白酶L具有37%的氨基酸同一性。针对C1.8编码的肽段产生的抗血清在免疫印迹上识别出一个28 kDa的滋养体蛋白。在Northern分析中，C1.8主要与环状体（滋养体阶段之前的生命周期阶段）的RNA杂交。综上所述，这些结果强烈表明我们已分离和鉴定的恶性疟原虫半胱氨酸蛋白酶基因编码TCP。
