Selection of recombinant anti‐SH3 domain antibodies by high‐throughput phage display

Abstract

Sachdev Sidhu is the recipient of the Protein Society 2015 Christian B. Anfinsen Award.Abstract: Antibodies are indispensable tools in biochemical research and play an expanding role as therapeutics. While hybridoma technology is the dominant method for antibody production, phage display is an emerging technology. Here, we developed and employed a high-throughput pipeline that enables selection of antibodies against hundreds of antigens in parallel. Binding selections using a phage-displayed synthetic antigen-binding fragment (Fab) library against 110 human SH3 domains yielded hundreds of Fabs targeting 58 antigens. Affinity assays demonstrated that representative Fabs bind tightly and specifically to their targets. Furthermore, we developed an efficient affinity maturation strategy adaptable to high-throughput, which increased affinity dramatically but did not compromise specificity. Finally, we tested Fabs in common cell biology applications and confirmed recognition of the full-length antigen in immunoprecipitation, immunoblotting and immunofluorescence assays. In summary, we have established a rapid and robust high-throughput methodology that can be applied to generate highly functional and renewable antibodies targeting protein domains on a proteome-wide scale.

A high-throughput pipeline for the production of synthetic antibodies for analysis of ribonucleoprotein complexes

“…5). Moreover, this success rate for antibody production is comparable with that achieved when high-throughput selections are carried out against well-characterized protein domains as antigens (e.g., SH3 domains) (Huang et al 2015). In addition to validating our streamlined method for screening for individual antigenbinding Fabs, this success rate validates our computational approach for choosing predicted structured regions that lie outside of annotated domains as a highly effective method for designing antigens for synthetic antibody selection.…”

“…In particular, we wanted to select regions that are likely to fold independently into stable structures, since such regions are required to optimize the chances of yielding antibodies by phage display approaches (Hornsby et al 2015). First, we searched for annotated domains since these have served as effective antigens for synthetic antibody production in other studies (Colwill and Graslund 2011;Huang et al 2015). However, as described above, we wanted to avoid choosing canonical RNA-binding domains as antigens in order to minimize the chances of producing antibodies that might interfere with RBP-RNA interactions.…”

“…In addition, we note that anti-STAU 2A5, one of the "moderate" Fabs, has been used successfully to identify STAU target mRNAs in RIP-Chip experiments (Laver et al 2013). Moreover, when necessary, weaker Fabs can be affinity matured to improve their performance in IPs (Li et al 2009;Huang et al 2015) and/or can be converted to bivalent IgGs that exhibit enhanced effective affinities due to avidity effects. In summary, our success in the design and expression of soluble antigens, many of which map outside of annotated domains (96%), and in obtaining one or more Fabs against a large majority of the antigens screened (66%) combined with the high success rate of our Fabs in IP of their of endogenous target proteins (89%), demonstrates the utility of our high-throughput pipeline for generating synthetic antibodies as tools for studies of RNPs.…”

“…For instance, given the recombinant nature of synthetic antibodies, they can be engineered or tagged in a variety of ways, and they represent an inexhaustible resource. In addition, through subsequent mutagenesis of a particular Fab and additional selections, higher affinity antibodies against a particular protein can be produced in a process referred to as affinity maturation (Li et al 2009;Huang et al 2015). Finally, synthetic antibodies are not associated with the ethical issues related to the use of antibodies raised in animals.…”

“…1). Importantly, our method incorporates several novel aspects compared with other high-throughput synthetic antibody production methodologies (Schofield et al 2007;Hornsby et al 2015;Huang et al 2015). These include (i) a computationally aided approach for designing effective antigens that lie outside of annotated domains, (ii) improved protocols for high-throughput antigen expression and purification, and (iii) a streamlined strategy to screen for individual antibodies and subclone them for expression in E. coli.…”