Zhang Di, Tözsér József, Waugh David S
Macromolecular Crystallography Laboratory, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD 21702-1201, USA.
Protein Expr Purif. 2009 Mar;64(1):89-97. doi: 10.1016/j.pep.2008.10.013. Epub 2008 Oct 30.
Alphaviruses cause serious diseases that pose a potential health threat to both humans and livestock. The nonstructural protein 2 (nsp2) encoded by alphaviruses is a multifunctional enzyme that is essential for viral replication and maturation. Its 39-kDa C-terminal domain (nsp2pro) is a cysteine protease that is responsible for cleaving a viral polyprotein at three sites to generate nonstructural proteins 1, 2, 3 and 4. In the present study, we evaluated nsp2pro domains from the following three sources as reagents for site-specific cleavage of fusion proteins: Venezuelan Equine Encephalitis Virus (VEEV), Semliki Forest Virus (SFV) and Sindbis Virus (SIN). All three alphavirus proteases cleaved model fusion protein substrates with high specificity but they were much less efficient enzymes than potyviral proteases from tobacco etch virus (TEV) and tobacco vein mottling virus (TVMV). Oligopeptide substrates were also cleaved with very low efficiency by the alphavirus proteases. We conclude that, in general, alphavirus nsp2pro proteases are not very useful tools for the removal of affinity tags from recombinant proteins although they do remain promising therapeutic targets for the treatment of a variety of diseases.
甲病毒可引发严重疾病，对人类和牲畜的健康构成潜在威胁。甲病毒编码的非结构蛋白2（nsp2）是一种多功能酶，对病毒复制和成熟至关重要。其39 kDa的C末端结构域（nsp2pro）是一种半胱氨酸蛋白酶，负责在三个位点切割病毒多聚蛋白，以产生非结构蛋白1、2、3和4。在本研究中，我们评估了来自以下三种来源的nsp2pro结构域作为融合蛋白位点特异性切割试剂的效果：委内瑞拉马脑炎病毒（VEEV）、Semliki森林病毒（SFV）和辛德毕斯病毒（SIN）。所有这三种甲病毒蛋白酶都能高度特异性地切割模型融合蛋白底物，但与烟草蚀纹病毒（TEV）和烟草脉斑驳病毒（TVMV）的马铃薯Y病毒蛋白酶相比，它们的酶活性要低得多。甲病毒蛋白酶对寡肽底物的切割效率也非常低。我们得出结论，一般而言甲病毒nsp2pro蛋白酶并非从重组蛋白中去除亲和标签的非常有用的工具，尽管它们仍是治疗多种疾病的有前景的治疗靶点。
