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nature biotechnology VOLUME 19 MAY 2001 http://biotech.nature.com RESEARCH ARTICLE
 

Summary: nature biotechnology ˇ VOLUME 19 ˇ MAY 2001 ˇ http://biotech.nature.com
RESEARCH ARTICLE
456
Libraries of hybrid proteins from distantly
related sequences
Volker Sieber, Carlos A. Martinez, and Frances H. Arnold*
We introduce a method for sequence homology­independent protein recombination (SHIPREC) that can cre-
ate 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.
Recombination is an important mechanism for the acquisition of
novel function in proteins. Various methods for using recombina-
tion in the design of protein libraries for laboratory evolution have
been described1­6. The majority of these "DNA-shuffling" methods
can recombine only closely related sequences (more than 70%

  

Source: Arnold, Frances H. - Division of Chemistry and Chemical Engineering, California Institute of Technology

 

Collections: Chemistry; Biology and Medicine