A COMBINATORIAL LIBRARY OF AN ALPHA-HELICAL BACTERIAL RECEPTOR DOMAIN
A COMBINATORIAL LIBRARY OF AN ALPHA-HELICAL BACTERIAL RECEPTOR DOMAIN
复制标题
DOI：
10.1093/protein/8.6.601
复制
发表时间：
1995-06-01
期刊：
影响因子：
--
通讯作者：
NYGREN, PA
中科院分区：
文献类型：
--
作者：
NORD, K;NILSSON, J;NYGREN, PA
描述了细菌受体衍生的α-螺旋结构域的溶剂暴露表面的组合文库的构建和特征。使用一种新的固相方法，利用含有对基因片段的杂色片段进行多次随机替换的单链寡核苷酸，以定向和连续的方式组装文库。在一个跨越81个核苷酸的区域中，13个残基同时替代了所有20个可能的氨基酸。氨基酸的密码子被模拟为在由葡萄球菌蛋白A的单价Fc结合域的三个α-螺旋中的两个组成的表面上进行随机化。在将PCR扩增的文库克隆到适合于展示突变体的噬菌体载体后，DNA测序分析表明，在突变的位置密码子随机分布。将该文库中的4个成员与链球菌G蛋白的白蛋白亲和标签进行融合，经人血清白蛋白亲和层析纯化后，用十二烷基硫酸钠电泳法、圆二色谱和生物传感器分析对融合蛋白进行了鉴定。分析表明，突变的蛋白A衍生物均能以可溶性全长蛋白的形式分泌。此外，圆二色谱分析表明，除了一个在螺旋2中引入了脯氨酸的突变体外，所有突变体都具有与野生型结构域非常一致的二级结构。这些结果证明，该α-螺旋受体文库的成员在暴露于溶剂的表面具有多个取代基，在大肠杆菌中保持稳定和可溶。讨论了将该文库用于表型选择策略以获得具有新功能的人工抗体的可能性。
The construction and characterization of a combinatorial library of a solvent-exposed surface of an alpha-helical domain derived from a bacterial receptor is described. Using a novel solid-phase approach, the library was assembled in a directed and successive manner utilizing single-stranded oligonucleotides containing multiple random substitutions for the variegated segments of the gene fragment. The simultaneous substitution of 13 residues to all 20 possible amino acids was carried out in a region spanning 81 nucleotides. The randomization was made in codons for amino acids that were modelled to be solvent accessible at a surface made up from two of the three alpha-helices of a monovalent Fc-binding domain of staphylococcal protein A. After cloning of the PCR-amplified library into a phagemid vector adapted for phage display of the mutants, DNA sequencing analysis suggested a random distribution of codons in the mutagenized positions. Four members of the library with multiple substitutions were produced in Escherichia coli as fusions to an albumin-binding affinity tag derived from streptococcal protein G. The fusion proteins were purified by human serum albumin affinity chromatography and subsequently characterized by SDS-electrophoresis, CD spectroscopy and biosensor analysis. The analyses showed that the mutant protein A derivatives could all be secreted as soluble full-length proteins. Furthermore, the CD analysis showed that all mutants, except one with a proline introduced into helix 2, have secondary structures in close agreement with the wild-type domain. These results proved that members of this alpha-helical receptor library with multiple substitutions in the solvent-exposed surface remain stable and soluble in E.coli. The possibility of using this library for a phenotypic selection strategy to obtain artificial antibodies with novel functions is discussed.