title: In vitro selection and characterization of a stable subdomain of phosphoribosylanthranilate isomerase.
abstract: The (beta(alpha))8-barrel is the most common enzyme fold and it is capable of catalyzing an enormous diversity of reactions. It follows that this scaffold should be an ideal starting point for engineering novel enzymes by directed evolution. However, experiments to date have utilized in vivo screens or selections and the compatibility of (beta(alpha))8-barrels with in vitro selection methods remains largely untested. We have investigated plasmid display as a suitable in vitro format by engineering a variant of phosphoribosylanthranilate isomerase (PRAI) that carried the FLAG epitope in active-site-forming loop 6. Trial enrichments for binding of mAb M2 (a mAb to FLAG) demonstrated that FLAG-PRAI could be identified from a 10(6)-fold excess of a FLAG-negative competitor in three rounds of in vitro selection. These results suggest PRAI as a useful scaffold for epitope and peptide grafting experiments. Further, we constructed a FLAG-PRAI loop library of approximately 10(7) clones, in which the epitope residues most critical for binding mAb M2 were randomized. Four rounds of selection for antibody binding identified and enriched for a variant in which a single nucleotide insertion produced a truncated (beta(alpha))8-barrel consisting of (beta(alpha))1-5beta6. Biophysical characterization of this clone, trPRAI, demonstrated that it was selected because of a 21-fold increase in mAb M2 affinity compared with full-length FLAG-PRAI. Remarkably, this truncated barrel was found to be soluble, structured, thermostable and monomeric, implying that it represents a genuine subdomain of PRAI and providing further evidence that such subdomains have played an important role in the evolution of the (beta(alpha))8-barrel fold.
authors: Department of Biochemistry, University of Cambridge, UK.
authors: Wayne M
authors: WM
authors: Patrick
authors: Jonathan M
authors: JM
authors: Blackburn
mesh_terms: D019747:Aldose-Ketose Isomerases / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D002942:Circular Dichroism; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013696:Temperature
chemical_list: D000939:Epitopes; D019747:Aldose-Ketose Isomerases; C029865:phosphoribosylanthranilate isomerase