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

Nan Zhong

2015

Scholarcy highlights

  • Antibodies are widely used in the scientific community, as therapeutic agents and research tools
  • We first compared the efficiencies of Fab selection using six different antigens appended with three different tags each or Streptavidin-binding peptide
  • The optimized process increased the number of antigens produced, the yields of antigen, Fab, and IgG materials
  • The process generated Fabs and IgGs shown to work in cell biology applications, such as immunoprecipitation and chromatin immunoprecipitation
  • We found that the promoter from pCWOri+ gave the highest yields, and used this information to design a new Fab expression vector, pSFV4. pSFV4 was tailor-made to optimize the transfer of the Fab sequences from the phagemid, in the originating library independent fashion, to the expression plasmid with a minimal amount of changes
  • The one third of the Fabs that did not perform well either recognized an epitope that is masked in the full-length protein or targeted an antigen that was not expressed in HEK293 cells
  • A) Scatter plot of a single point competitive ELISA data obtained for BAZ2B, BRD4, CBX3, CBX5, EP300, JMJD2A, JMJD2C, JMJD3, L3MBTL2, PHF8, PRDM4, SFMBT2, SMARCA4 where 96 individual Fab-phage clones for each of the targets were tested in high-throughput format

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