Kamei Ayako, Hauser Paul S, Beckstead Jennifer A, Weers Paul M M, Ryan Robert O
Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr Way, Oakland, CA 94609, United States.
Protein Expr Purif. 2012 Jun;83(2):113-6. doi: 10.1016/j.pep.2012.03.014. Epub 2012 Apr 1.
Expressed protein ligation (EPL) was performed to investigate sequence requirements for a variant human apolipoprotein A-I (apoA-I) to adopt a folded structure. A C-terminal truncated apoA-I, corresponding to residues 1-172, was expressed and isolated from Escherichia coli. Compared to full length apoA-I (243 amino acids), apoA-I(1-172) displayed less α-helix secondary structure and lower stability in solution. To determine if extension of this polypeptide would confer secondary structure content and/or stability, 20 residues were added to the C-terminus of apoA-I(1-172) by EPL, creating apoA-I(Milano)(1-192). The EPL product displayed biophysical properties similar to full-length apoA-I(Milano). The results provide a general protein engineering strategy to modify the length of a recombinant template polypeptide using synthetic peptides as well as a convenient, cost effective way to investigate the structure/function relations in apolipoprotein fragments or domains of different size.
进行表达蛋白连接（EPL）以研究变异型人载脂蛋白A-I（apoA-I）形成折叠结构的序列要求。从大肠杆菌中表达并分离出对应于1-172位残基的C末端截短的apoA-I。与全长apoA-I（243个氨基酸）相比，apoA-I(1-172)在溶液中显示出较少的α-螺旋二级结构和较低的稳定性。为了确定该多肽的延伸是否会赋予二级结构含量和/或稳定性，通过EPL在apoA-I(1-172)的C末端添加了20个残基，从而产生了apoA-I（米兰）(1-192)。EPL产物显示出与全长apoA-I（米兰）相似的生物物理性质。这些结果提供了一种通用的蛋白质工程策略，即使用合成肽来修饰重组模板多肽的长度，以及一种方便、经济有效的方法来研究不同大小的载脂蛋白片段或结构域中的结构/功能关系。
