Jagadish M N, Vaughan P R, Irving R A, Azad A A, Macreadie I G
CSIRO, Division of Biomolecular Engineering, Parkville Laboratory, Victoria, Australia.
Gene. 1990 Nov 15;95(2):179-86. doi: 10.1016/0378-1119(90)90360-4.
Various expression vectors containing a cDNA fragment encoding all but the first five amino acids (aa) of the large polyprotein (N-VP2-VP4-VP3-C) of infectious bursal disease virus were transformed into yeasts. In both Saccharomyces cerevisiae and Schizosaccharomyces pombe, co- or post-translational processing of the unfused large polyprotein occurred, generating a stable C-terminal product (VP3) or correct size, but without any detectable N-terminal product (VP2). Furthermore, when the processing of the polyprotein was interrupted, because of an engineered in-frame site-specific insertion of 4 aa, even VP3 (as part of the unprocessed polyprotein) was undetected. VP2 was detected in S. cerevisiae only when fused to yeast pre-sequences at the N terminus, suggesting that in yeast, VP2 or the unprocessed polyprotein, in the absence of its native N terminus or proper protection of its N-terminal aa residues is susceptible to proteolytic degradation. The first 8 aa of a modified pre-sequence of the CUP1 gene product and the pre-pro sequence of MF alpha 1 gene product have been used for stable intra- and extra-cellular production of VP2, respectively.
将含有传染性法氏囊病病毒大聚蛋白（N-VP2-VP4-VP3-C）除前五个氨基酸（aa）外的编码cDNA片段的各种表达载体转化到酵母中。在酿酒酵母和粟酒裂殖酵母中，未融合的大聚蛋白发生了共翻译或翻译后加工，产生了稳定的C端产物（VP3）或正确大小的产物，但未检测到任何N端产物（VP2）。此外，当由于工程化的框内4个氨基酸的位点特异性插入而中断聚蛋白的加工时，甚至VP3（作为未加工聚蛋白的一部分）也未被检测到。只有当VP2在N端与酵母前导序列融合时，才在酿酒酵母中检测到，这表明在酵母中，VP2或未加工的聚蛋白，在没有其天然N端或其N端氨基酸残基没有得到适当保护的情况下，易受蛋白水解降解。CUP1基因产物修饰前导序列的前8个氨基酸和MF alpha 1基因产物的前原序列已分别用于VP2在细胞内和细胞外的稳定产生。
