In vitro selection and characterization of a stable subdomain of phosphoribosylanthranilate isomerase

Abstract

The (βα)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 (βα)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 106‐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 ≈ 107 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 (βα)8‐barrel consisting of (βα)1−5β6. 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 (βα)8‐barrel fold.

The Structure of a Truncated Phosphoribosylanthranilate Isomerase Suggests a Unified Model for Evolution of the (βα)8 Barrel Fold

“…Previously, this sequence was subcloned with an initiator methionine, a C-terminal His 6 tag and a threeresidue linker (Ala-Gly-Ser) between trPRAI and the His 6 tag. 25 We expressed this C-terminally His 6 -tagged version of trPRAI in E. coli and purified the recombinant protein using nickel affinity and sizeexclusion chromatography (SEC). The symmetrical gel-filtration profile of trPRAI at two concentrations indicates a monodisperse species, and multi-angle laser light-scattering (MALLS) data show that the protein is monomeric in solution (Fig.…”

“…25 Unlike other part-barrel subdomains, trPRAI is readily purified from the soluble cell lysate. Here, we report that trPRAI is monomeric and well ordered in solution, and our NMR-derived solution structure of trPRAI demonstrates unequivocally that three-quarter barrels can indeed form native-like structures in solution.…”

A Study in Molecular Contingency: Glutamine Phosphoribosylpyrophosphate Amidotransferase is a Promiscuous and Evolvable Phosphoribosylanthranilate Isomerase

“…The construction of pMS401, for the expression of (His) 6 -tagged E. coli PRAI, was described previously. 55 The E. coli trpC gene, encoding residues 1-259 of the bifunctional IGPS:PRAI monomer, 21 was amplified from the ASKA clone pCA24N-trpCF using primers trpC_NcoI.for and trpC_BamHI.rev. These oligonucleotides also encoded restriction sites, enabling the digestion of the PCR product with NcoI and BamHI and its ligation with pMS401 (which had been treated with the same enzymes to excise trpF).…”

A plasmid display platform for the selection of peptides exhibiting a functional cell‐penetrating phenotype

“…Previously, it has been shown that selections with the PD system could be performed in the Escherichia coli cytoplasmic milieu without purification. 21,24 It was not clear if the contaminating prokaryotic material would adversely affect the transfection of mammalian cells.…”