General method for sequence-independent site-directed chimeragenesis.
We have developed a simple and general method that allows for the facile recombination of distantly related (or unrelated) proteins at multiple discrete sites. To evaluate the sequence-independent site-directed chimeragenesis (SISDC) method, we have recombined beta-lactamases TEM-1 and PSE-4 at seven sites, examined the quality of the chimeric genes created, and screened the library of 2(8) (256) chimeras for functional enzymes. Probe hybridization and sequencing analyses revealed that SISDC generated a random library with little sequence bias and in which all targeted fragments were recombined in the desired order. Sequencing the genes from clones having functional lactamases identified 14 unique chimeras. These chimeras are characterized by a lower level of disruption, as calculated by the SCHEMA algorithm, than the library as a whole. These results illustrate the use of SISDC in creating designed chimeric protein libraries and further illustrate the ability of SCHEMA to identify chimeras whose folded structures are likely not to be disrupted by recombination.
Hiraga
Kaori
K
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
Arnold
Frances H
FH
D000465:Algorithms; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002678:Chimera / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D019020:Directed Molecular Evolution / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D019151:Peptide Library; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D011995:Recombination, Genetic; D001618:beta-Lactamases / Q000235:genetics
D004274:DNA, Recombinant; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; C410213:beta-lactamase PSE-4; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1