Libraries of hybrid proteins from distantly related sequences
- 1 May 2001
- journal article
- research article
- Published by Springer Nature in Nature Biotechnology
- Vol. 19 (5) , 456-460
- https://doi.org/10.1038/88129
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.Keywords
This publication has 23 references indexed in Scilit:
- Recombination and chimeragenesis by in vitro heteroduplex formation and in vivo repairNucleic Acids Research, 1999
- A simple in vivo assay for increased protein solubilityProtein Science, 1999
- Design of a Novel P450: A Functional Bacterial−Human Cytochrome P450 ChimeraBiochemistry, 1998
- Molecular evolution by staggered extension process (StEP) in vitro recombinationNature Biotechnology, 1998
- Drug metabolism by Escherichia coli expressing human cytochromes P450Nature Biotechnology, 1997
- Recombinant Human Cytochrome P450 1A2 and an N-Terminal-Truncated Form: Construction, Purification, Aggregation Properties, and Interactions with Flavodoxin, Ferredoxin, and NADPH-Cytochrome P450 ReductaseArchives of Biochemistry and Biophysics, 1996
- A novel structural basis for membrane association of a protein: Construction of a chimeric soluble mutant of (S)-mandelate dehydrogenase from Pseudomonas putidaBiochemistry, 1993
- Chloramphenicol AcetyltransferaseAnnual Review of Biophysics, 1991
- Cloning and Isolation of Human Cytochrome P-450 cDNAs Homologous to Dioxin-inducible Rabbit mRNAs Encoding P-450 4 and P-450 6DNA, 1985
- Partial characterization of a barbiturate-induced cytochrome P-450-dependent fatty acid monooxygenase from BacillusmegateriumBiochemical and Biophysical Research Communications, 1983