Optimizing Production of Antigens and Fabs in the Context of Generating Recombinant Antibodies to Human Proteins

Abstract

We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling. Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that in vivo biotinylation through the use of an Avi tag was the most productive method. Phage display selections were performed on 265 in vivo biotinylated antigen domains. High-affinity Fabs (<20nM) were obtained for 196. We constructed and optimized a new expression vector to produce in vivo biotinylated Fabs in E. coli. This increased average yields up to 10-fold, with an average yield of 4 mg/L. For 118 antigens, we identified Fabs that could immunoprecipitate their full-length endogenous targets from mammalian cell lysates. One Fab for each antigen was converted to a recombinant IgG and produced in mammalian cells, with an average yield of 15 mg/L. In summary, we have optimized each step of the pipeline to produce recombinant antibodies, significantly increasing both efficiency and yield, and also showed that these Fabs and IgGs can be generally useful for chromatin immunoprecipitation (ChIP) protocols.

Neutrophil dysregulation is pathogenic in idiopathic inflammatory myopathies

“…JN792439), which contains an N-terminal His 6 -tag and a C-terminal Avi-tag. Expression and purification were performed as described previously (39,40). Biotinylated MDA5 and HisRS were produced by coexpressing the generated constructs with the bacterial BirA biotin ligase in E. coli strain BL21(DE3) R3.…”

The path to VICTORy – a beginner's guide to success using commercial research antibodies

“…This gap also emphasizes the lack of a reliable and independent source of validated standard target proteins (Gilda et al, 2015;Quarmby et al, 1998). Given that there is a market for nearly 4×10 6 cr-Abs, it cannot be beyond human ingenuity to set up an international Public Interest Entity, which, on a not-for-profit basis, produces precisely defined proteomic targets, for example the principal components of the human and mouse proteomes, and verifies antibodies that recognize them in 'pillar' validation technologies (Colwill et al, 2011;Edwards et al, 2017;Zhong et al, 2015;Weller, 2016). These antibodies and target standards could then be certified and maintained as standard open-source reference pairs, routinely available under commercial terms, to help validate and align the specificity of other cr-Abs.…”

Biotin-tagged proteins: Reagents for efficient ELISA-based serodiagnosis and phage display-based affinity selection

“…Over the last two decades, specific and efficient capture of biotin-tagged proteins on streptavidin surface by virtue of extremely high-affinity interaction has found use in a diverse range of biological applications [ 1 , 2 ] including immunoassays [ 3 – 6 ], phage display-based affinity selection [ 7 , 8 ], and affinity-based purification [ 9 , 10 ]. Conventional approaches for immobilization of proteins involve their passive adsorption on polystyrene surfaces.…”