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Libraries of hybrid proteins from distantly related sequences


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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Figure 1: SHIPREC procedure.
Figure 2: Nucleotide and amino acid sequences in the region of the crossover of functional hybrid cytochrome P450s RC1 and RC2.


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V.S. was supported by a research fellowship from the Deutsche Forschungsgemeinschaft (DGF). This work was funded by Maxygen Inc.

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Correspondence to Frances H. Arnold.

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Sieber, V., Martinez, C. & Arnold, F. Libraries of hybrid proteins from distantly related sequences. Nat Biotechnol 19, 456–460 (2001).

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