title: Libraries of hybrid proteins from distantly related sequences.
abstract: 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.
authors.affiliation: Division of Chemistry and Chemical Engineering 210-41, California Institute of Technology, Pasadena CA 91105, USA.
authors.forename: V
authors.initials: V
authors.lastname: Sieber
authors.forename: C A
authors.initials: CA
authors.lastname: Martinez
authors.forename: F H
authors.initials: FH
authors.lastname: Arnold
mesh_terms: D000595:Amino Acid Sequence; D019677:Artificial Gene Fusion; D001426:Bacterial Proteins; D001483:Base Sequence; D019388:Cytochrome P-450 CYP1A2 / Q000737:chemistry* / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism; D015723:Gene Library; D006801:Humans; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D009251:NADPH-Ferrihemoprotein Reductase; D015202:Protein Engineering / Q000379:methods; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics*; D017385:Sequence Homology; D012995:Solubility
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D019388:Cytochrome P-450 CYP1A2; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases