Generation of variability by in vivo recombination of halves of a (beta/alpha)8 barrel protein.
Similar to what has been achieved with nucleic acids, directed evolution of proteins would be greatly facilitated by the availability of large libraries and efficient selection methods. So far, host cell transformation efficiency has been a bottleneck, practically limiting libraries to sizes less than 10(9). One way to circumvent this problem has been implemented with antibody systems, where contribution to the binding site is provided by two different polypeptides (light and heavy chains). The central concept is the construction of binary systems in which the gene from the two chains are separated by a cre-lox recombinase recognition site, packaged in a phage, and subsequently introduced, by multiple infection, into a recombinase expressing cell [Sblattero D, Bradbury A. Nat Biotechnol 2000;18(1):75-80]. Here, we describe the development of a system which applies the same concept to a single-domain enzyme, the cytoplasmic (beta/alpha)8 barrel protein phosphoribosyl anthranilate isomerase (PRAI) from E. coli. For that purpose, we identified the site at which a loop containing the recognition sequence for cre-lox recombinase could be inserted yielding a functional enzyme. We evaluated the effect of this insertion on the capability of the engineered gene to complement a trp F-E. coli strain and the efficiency of the system to recover the original sequence from an abundance of non-functional mutant genes.
Saab-Rincón
Gloria
G
Instituto de Biotecnología, UNAM, Apartado Postal 510-3, Cuernavaca, Morelos 62271, México.
Mancera
Eugenio
E
Montero-Morán
Gabriela
G
Sánchez
Filiberto
F
Soberón
Xavier
X
D019747:Aldose-Ketose Isomerases / Q000737:chemistry; D001483:Base Sequence; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D019143:Evolution, Molecular; D015723:Gene Library; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D008957:Models, Genetic; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D017433:Protein Structure, Secondary; D011995:Recombination, Genetic; D013997:Time Factors
D009841:Oligonucleotides; D010455:Peptides; D019747:Aldose-Ketose Isomerases; C029865:phosphoribosylanthranilate isomerase