Biosynthetic phage display: a novel protein engineering tool combining chemical and genetic diversity
Biosynthetic phage display: a novel protein engineering tool combining chemical and genetic diversity
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DOI：
10.1016/s1074-5521(00)00102-2
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发表时间：
2000-04-01
影响因子：
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通讯作者：
Kossiakoff, A
中科院分区：
文献类型：
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作者：
Dwyer, MA;Lu, WY;Kossiakoff, A
背景：自然界中的分子多样性是由遗传和化学元素共同作用形成的。我们已经开发出一种方法，允许在一个蛋白质工程工具中选择性地操作这两个元件。它结合了通过天然化学连接将非天然氨基酸引入蛋白质的能力和通过噬菌体展示突变对蛋白质进行穷尽定向突变的能力。结果：构建了具有全功能的蛋白酶抑制剂Eglin c的生物合成版本。氨基末端片段(8-40位)由非天然氨基酸化学合成，其余的羧基末端片段以噬菌体展示突变的形式在丝状噬菌体上表达为gIIIp或gVIIIp的30个氨基酸残基延伸。天然的化学连接被用来连接这两个片段，并产生了一种可重新折叠为其活性形式的蛋白质。为了便于引入的非天然氨基酸的包装，通过噬菌体展示突变完全随机化了羧基末端片段中的52和54位残基。虽然观察到的52和54位氨基酸的大多数溶液是疏水的，补充了引入的非天然氨基酸的立体化学，但在这些位置观察到了大量的残基(由于立体化学和电荷标准而意想不到)。结论：多肽合成和噬菌体展示突变相结合可以产生一种非常强大的蛋白质工程工具。通过使用噬菌体展示诱变技术评估一组目标部位的所有可能的氨基酸类型组合，可以选择所引入的非天然残留物周围环境的物理性质。
Background: Molecular diversity in nature is developed through a combination of genetic and chemical elements. We have developed a method that permits selective manipulation of both these elements in one protein engineering tool. It combines the ability to introduce non-natural amino acids into a protein using native chemical ligation with exhaustive targeted mutagenesis of the protein via phage-display mutagenesis.Results: A fully functional biosynthetic version of the protease inhibitor eglin c was constructed. The amino-terminal fragment (residues 8-40) was chemically synthesized with a non-natural amino acid at position 25, The remaining carboxy-terminal fragment was expressed as a 30-residue peptide extension of gIIIp or gVIIIp on filamentous phage in a phage-display mutagenesis format. Native chemical ligation was used to couple the two fragments and produced a protein that refolded to its active form. To facilitate the packing of the introduced non-natural amino acid, residues 52 and 54 in the carboxy-terminal fragment were fully randomized by phage-display mutagenesis. Although the majority of the observed solutions for residues 52 and 54 were hydrophobic complementing the stereochemistry of the introduced non-natural amino acid a significant number of residues (unexpected because of stereochemical and charge criteria) were observed in these positions.Conclusions: Peptide synthesis and phage-display mutagenesis can be combined to produce a very powerful protein engineering tool. The physical properties of the environment surrounding the introduced non-natural residue can be selected for by evaluating all possible combinations of amino acid types at a targeted set of sites using phage-display mutagenesis.