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

Zhong N, Loppnau P, Seitova A, Ravichandran M, Fenner M, Jain H, Bhattacharya A, Hutchinson A, Paduch M, Lu V, Olszewski M, Kossiakoff AA, Dowdell E, Koide A, Koide S, Huang H, Nadeem V, Sidhu SS, Greenblatt JF, Marcon E, Arrowsmith CH, Edwards AM, Gräslund S - PLoS ONE (2015)

Bottom Line: We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling.This increased average yields up to 10-fold, with an average yield of 4 mg/L.One Fab for each antigen was converted to a recombinant IgG and produced in mammalian cells, with an average yield of 15 mg/L.

View Article: PubMed Central - PubMed

Affiliation: Structural Genomics Consortium, University of Toronto, MaRS South tower, 101 College street, Toronto, ON M5G 1L7, Canada.

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.

No MeSH data available.


Performance consistency among Fabs and IgGs generated against the same target.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.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4593582&req=5

pone.0139695.g003: Performance consistency among Fabs and IgGs generated against the same target.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.

Mentions: We analyzed 811 Fabs (for 186 antigens) by one or both of these methods. 407 unambiguously immunoprecipitated their cognate antigens from cell lysates. These 407 Fabs corresponded to 118 antigens, or 63% of the antigens tested (Table 1). We also observed that Fabs and IgGs produced from the same phagemid exhibited similar immunoprecipitation efficiencies and batch-to-batch variability was negligible (Fig 3). These results suggest that recombinant Fab antibodies are effective in recognizing their cognate antigens in cell lysates. Many of these reagents also proved suitable for immunofluorescence (IF) [25].


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

Zhong N, Loppnau P, Seitova A, Ravichandran M, Fenner M, Jain H, Bhattacharya A, Hutchinson A, Paduch M, Lu V, Olszewski M, Kossiakoff AA, Dowdell E, Koide A, Koide S, Huang H, Nadeem V, Sidhu SS, Greenblatt JF, Marcon E, Arrowsmith CH, Edwards AM, Gräslund S - PLoS ONE (2015)

Performance consistency among Fabs and IgGs generated against the same target.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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4593582&req=5

pone.0139695.g003: Performance consistency among Fabs and IgGs generated against the same target.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.
Mentions: We analyzed 811 Fabs (for 186 antigens) by one or both of these methods. 407 unambiguously immunoprecipitated their cognate antigens from cell lysates. These 407 Fabs corresponded to 118 antigens, or 63% of the antigens tested (Table 1). We also observed that Fabs and IgGs produced from the same phagemid exhibited similar immunoprecipitation efficiencies and batch-to-batch variability was negligible (Fig 3). These results suggest that recombinant Fab antibodies are effective in recognizing their cognate antigens in cell lysates. Many of these reagents also proved suitable for immunofluorescence (IF) [25].

Bottom Line: We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling.This increased average yields up to 10-fold, with an average yield of 4 mg/L.One Fab for each antigen was converted to a recombinant IgG and produced in mammalian cells, with an average yield of 15 mg/L.

View Article: PubMed Central - PubMed

Affiliation: Structural Genomics Consortium, University of Toronto, MaRS South tower, 101 College street, Toronto, ON M5G 1L7, Canada.

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.

No MeSH data available.