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

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

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

<div><p>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 <i>in vivo</i> biotinylation through the use of an Avi tag was the most productive method. Phage display selections were performed on 265 <i>in vivo</i> biotinylated antigen domains. High-affinity Fabs (<20nM) were obtained for 196. We constructed and optimized a new expression vector to produce <i>in vivo</i> biotinylated Fabs in <i>E</i>. <i>coli</i>. 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.</p></div

Performance consistency among Fabs and IgGs generated against the same target.

<p>Multiple Fabs and IgGs against several targets were used to immunoprecipitate their corresponding FLAG-tagged antigens. Western blot was performed and the presence of the FLAG-tagged immunoprecipitated protein was detected with an antibody against the tag. A) CBX3. B) L3MBTl2, C) SFMBT2, D) TDRD3. FLAG-tagged GFP was used as control (data not shown). Fab batches are labeled with a trailer “-Bxxx” and IgG batches are labeled with a trailer “-IBxxx”. Fabs against CBX3 and SFMBT2 have been produced twice (CBX3 (B002, B004); SFMBT2 (B002, B004)) while Fabs against L3MBTL2 and TDRD3 have been produced only once (L3MBTL2 (B001); TDRD3 (B001)). Multiple IgGs have been produced with corresponding IB numbers. Fabs/IgGs derived from the same phagemid clone have similar efficiencies and show a high lot-to-lot consistency.</p

Fab and IgG production.

<p>(A) Comparison of purification yields between different expression vectors using an anti-MBP Fab as an example. The large-scale purification method on the ÄKTA Xpress including a heat denaturation step was used. (B) SDS-PAGE gel showing the anti-MBP Fab produced with various expression vectors and purified in triplicate. (C) IgG production yields with and without the dilution strategy.</p

Overview of targets and their success rates.

<p>Overview of the 265 antigen domains included in the study and what protein families they belong to. Family success rate was calculated as percentage of targets that were successful in selections and which also yielded at least one antibody that passed cell-based validation.</p><p>Overview of targets and their success rates.</p

Comparison of antigen immobilization methods.

<p>(A) Three different affinity tags were tested for antigen immobilization in phage display; <i>in vivo</i> biotinylation through an Avi tag, SBP and GST tags. The diversity of Fabs derived from these differentially tagged antigens was then compared. (B) Immunoprecipitation with Fabs selected against either Avi-tagged antigen or GST-tagged antigen from a cell lysate expressing FLAG-tagged target protein. Immunoprecipitated antigen was detected with an M2 antibody against the FLAG tag. Fabs selected against Avi-tagged antigen generally show a higher recovery of the antigen.</p

Assessment of a method to characterize antibody selectivity and specificity for use in immunoprecipitation

Antibodies are used in multiple cell biology applications, but there are no standardized methods to assess antibody quality—an absence that risks data integrity and reproducibility. We describe a mass spectrometry–based standard operating procedure for scoring immunoprecipitation antibody quality. We quantified the abundance of all the proteins in immunoprecipitates of 1,124 new recombinant antibodies for 152 chromatin-related human proteins by comparing normalized spectral abundance factors from the target antigen with those of all other proteins. We validated the performance of the standard operating procedure in blinded studies in five independent laboratories. Antibodies for which the target antigen or a member of its known protein complex was the most abundant protein were classified as 'IP gold standard'. This method generates quantitative outputs that can be stored and archived in public databases, and it represents a step toward a platform for community benchmarking of antibody quality