Libraries of hybrid proteins from distantly related sequences

被引：160

作者：

Sieber, V ^{[1
]}

Martinez, CA ^{[1
]}

Arnold, FH ^{[1
]}

机构：

[1] CALTECH, Div Chem & Chem Engn, Pasadena, CA 91105 USA

来源：

NATURE BIOTECHNOLOGY | 2001年 / 19卷 / 05期

关键词：

D O I：

10.1038/88129

中图分类号：

Q81 [生物工程学（生物技术）]; Q93 [微生物学];

学科分类号：

071005 ; 0836 ; 090102 ; 100705 ;

摘要：

We introduce a method for sequence homology-independent protein recombination (SHIPREC) that can create libraries of single-crossover hybrids of unrelated or distantly related proteins. The method maintains the proper sequence alignment between the parents and introduces crossovers mainly at structurally related sites distributed over the aligned sequences. We used SHIPREC to create a library of interspecies hybrids of a membrane-associated human cytochrome P450 (1A2) and the heme domain of a soluble bacterial P450 (BM3). By fusing the hybrid gene library to the gene for chloramphenicol acetyl transferase (CAT), we were able to select for soluble and properly folded protein variants. Screening for 1A2 activity (deethylation of 7-ethoxyresorufin) identified two functional P450 hybrids that were more soluble in the bacterial cytoplasm than the wild-type 1A2 enzyme.

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页码：456 / 460

页数：5

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