Alvarez Vanina, Parussini Fabiola, Aslund Lena, Cazzulo Juan J
Instituto de Investigaciones Biotecnológicas, Universidad Nacional de General San Martin, 1650 San Martin, Provincia de Buenos Aires, Argentina.
Protein Expr Purif. 2002 Dec;26(3):467-75. doi: 10.1016/s1046-5928(02)00565-x.
Cruzipain, the major cysteine proteinase from Trypanosoma cruzi, is a member of the papain family that contains a C-terminal domain in the mature enzyme, in addition to a catalytic moiety homologous to papain and some mammalian cathepsins. The native enzyme is expressed as a complex mixture of isoforms and has not been crystallized. Previous attempts to express recombinant mature cruzipain containing the C-terminal domain have failed. For this reason, the three-dimensional structure of the complete mature enzyme is not known, although the structure of a recombinant truncated molecule lacking the C-terminal domain (cruzaindeltac) has been determined. We report here the expression of active, N-glycosylated, complete mature cruzipain in an insect cell/baculovirus system. The purified recombinant enzyme, obtained with a yield of about 0.2 mg/100 ml of culture supernatant, has an apparent molecular mass similar, and an identical N-terminal sequence, compared with the native enzyme. The expressed protein is able to process itself by self-proteolysis, leaving the isolated C-terminal domain, and has kinetic properties similar to those of native cruzipain, although some differences in substrate specificity were found. These results open up the possibility of obtaining recombinant intact mature cruzipain of a quality and in quantity suitable for X-ray crystallography.
克氏锥虫蛋白酶（cruzipain）是克氏锥虫的主要半胱氨酸蛋白酶，属于木瓜蛋白酶家族成员。在成熟酶中，除了具有与木瓜蛋白酶和一些哺乳动物组织蛋白酶同源的催化部分外，还含有一个C末端结构域。天然酶以同工型的复杂混合物形式表达，尚未结晶。先前尝试表达含有C末端结构域的重组成熟克氏锥虫蛋白酶均告失败。因此，尽管缺乏C末端结构域的重组截短分子（cruzaindeltac）的结构已被确定，但完整成熟酶的三维结构仍不清楚。我们在此报告在昆虫细胞/杆状病毒系统中表达有活性的、N-糖基化的完整成熟克氏锥虫蛋白酶。纯化的重组酶产量约为0.2 mg/100 ml培养上清液，与天然酶相比，其表观分子量相似，N末端序列相同。表达的蛋白能够通过自我切割进行加工，留下分离的C末端结构域，并且具有与天然克氏锥虫蛋白酶相似的动力学特性，尽管在底物特异性方面发现了一些差异。这些结果为获得质量和数量适合X射线晶体学研究的重组完整成熟克氏锥虫蛋白酶开辟了可能性。
