Viaplana E, Feliu J X, Corchero J L, Villaverde A
Department de Genètica i Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, Spain.
Biochim Biophys Acta. 1997 Dec 5;1343(2):221-6. doi: 10.1016/s0167-4838(97)00114-3.
The VP60 capsid protein of rabbit haemorrhagic disease virus (60 kDa) has been fused to the C-terminus of beta-galactosidase and produced in E. coli from two related expression vectors. One of these vectors, carries a 429 bp DNA segment encoding the N-terminus peptide of VP60, and directs the synthesis of a larger fusion that contains the entire viral protein. Both fusion proteins are efficiently cleaved at a presumed trypsin-like target site within the carboxy moiety of beta-galactosidase (Arg 611-Thr 612), which is activated by the presence of the viral partner. In the larger fusion, VP60 is released by a cleavage within the linker region that affects about 10% of the chimeric proteins. In this situation, the resulting beta-galactosidase-like fragment recovers its natural proteolytic stability. These results prove that cryptic cleavage sites in beta-galactosidase can be efficiently activated in a fusion protein and suggest that this activation is based on reversible steric constraints generated by the fusion partner.
兔出血症病毒的VP60衣壳蛋白（60 kDa）已与β-半乳糖苷酶的C末端融合，并通过两个相关的表达载体在大肠杆菌中产生。其中一个载体携带一段429 bp的DNA片段，编码VP60的N末端肽，并指导合成包含整个病毒蛋白的更大融合蛋白。两种融合蛋白都在β-半乳糖苷酶羧基部分（Arg 611-Thr 612）内一个假定的类胰蛋白酶靶位点处有效切割，该位点因病毒伴侣的存在而被激活。在较大的融合蛋白中，VP60通过连接区的切割释放，约10%的嵌合蛋白受到影响。在这种情况下，产生的β-半乳糖苷酶样片段恢复了其天然的蛋白水解稳定性。这些结果证明，β-半乳糖苷酶中的隐蔽切割位点可以在融合蛋白中有效激活，并表明这种激活基于融合伴侣产生的可逆空间限制。
