Evolution of proteins with genetically encoded "chemical warheads"

Abstract

We recently developed a phage-based system for the evolution of proteins in bacteria with expanded amino acid genetic codes. Here we demonstrate that the unnatural amino acid p-boronophenylalanine (BF) confers a selective advantage in the evolution of glycan-binding proteins. We show that an unbiased library of naive antibodies with NNK-randomized V(H) CDR3 loops converges upon mutants containing BF when placed under selection for binding to a model acyclic amino sugar. This work represents a first step in the evolution of carbohydrate-binding proteins that use a reactive unnatural amino acid "warhead" and demonstrates that a "synthetic" genetic code can confer a selective advantage by increasing the number of functional groups available to evolution.

Figure 1

p-Boronophenylalanine can form boronate esters with diols.

Figure 2

(A) Binding of glucamine resin by phage clones. Mean binding of three unique clones containing BF from before selection (pre) is compared to mean binding of five unique clones containing BF from after the third round of selection (post) and binding of the two most frequent clones obtained from selection (172-6 and 172-7). Binding is reported as fraction eluted, determined by infection titers, multiplied by 10⁴. Error bars are ± std. dev. of triplicate experiments. (B) Binding of BSA and BSA-conjugated glycans by phage clones. Samples compared are a clone from before selection that does not contain a BF (13-1), two clones from before selection that each contains a BF (172-3 and 172-4), and the most frequently selected 172-6 and 172-7 phage clones. Binding is reported as fraction eluted, determined by infection titers, multiplied by 10⁵. Error bars are + std. dev. of triplicate experiments. (C) Binding of glucamine resin by expressed soluble Fabs. Eluted protein was coated onto wells of an ELISA plate, bound by an HRP conjugated antibody against human κ light chain, and detected using a fluorogenic substrate (QuantaBlu). Error bars represent ± std. dev. of triplicate experiments.