Role of capsid structure and membrane protein processing in determining the size and copy number of peptides displayed on the major coat protein of filamentous bacteriophage
Role of capsid structure and membrane protein processing in determining the size and copy number of peptides displayed on the major coat protein of filamentous bacteriophage
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DOI：
10.1006/jmbi.1996.0378
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发表时间：
1996-07-05
影响因子：
5.6
通讯作者：
Perham, RN
中科院分区：
文献类型：
--
作者：
Malik, P;Tarry, TD;Perham, RN
丝状噬菌体病毒粒子可经改造，在主要衣壳蛋白（pVIII）的所有2700个拷贝的N端区域展示小的外源肽段，但较大的肽段只能在杂合病毒粒子中容纳，在这种杂合病毒粒子中，修饰的和野生型衣壳蛋白亚基是相互交错的。一般认为，杂合病毒粒子中所接纳的肽段拷贝数与肽段大小有关：插入片段越大，组装后的病毒粒子中修饰的衣壳蛋白亚基数量越少。然而，我们在此表明，一些大的肽段能够以比小肽段高得多的拷贝数展示，并且一些相对较小的肽段在杂合病毒粒子中即使能展示，展示效果也很差。对一种重组病毒粒子的X射线衍射研究以及对具有已知结构的肽段和蛋白质表位进行的模型构建实验表明，容纳比在体内所能产生的大得多的结构且不干扰衣壳蛋白的包装是可行的。我们进一步表明，某些肽段的插入极大地减慢甚至阻止了大肠杆菌细胞内膜上的导肽酶对pVIII前衣壳的加工。在插入片段对前衣壳加工速率（病毒组装的关键步骤）的影响与随后组装的杂合病毒粒子中成熟但经修饰的蛋白质数量之间发现了良好的相关性。这些结果对基于丝状噬菌体的肽展示系统的设计具有重要意义。（C）1996学术出版社有限公司
Filamentous bacteriophage virions can be engineered to display small foreign peptides in the N-terminal regions of all 2700 copies of the major coat protein (pVIII), but larger peptides can be accommodated only in hybrid virions, in which modified and wild-type coat protein subunits are interspersed. The copy number of peptides accepted in hybrid virions is generally believed to be related to peptide size: the larger the insert, the lower the number of modified coat protein subunits in the assembled virion. However, we show here that some large peptides can be displayed at a much higher copy number than smaller ones and that some relatively small peptides are poorly displayed, if at all, in hybrid virions. X-ray diffraction studies of a recombinant virion together with model building experiments with peptide and protein epitopes of known structure demonstrated that it is feasible to accommodate much larger structures, without perturbation of the capsid protein packing, than it has proved possible to generate in vivo. We show further that the insertion of certain peptides greatly slowed or even prevented the processing of the pVIII pro-coat by leader peptidase at the inner membrane of the Escherichia coli cell. A good correlation was found between the effect of the insert on the rate of the processing of the pro-coat, an essential step in virus assembly, and the number of the mature but modified proteins in the subsequently assembled hybrid virion. These results have important implications for the design of peptide display systems based on filamentous bacteriophage. (C) 1996 Academic Press Limited