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Decoding Selection Bias Imparted by Unpaired Cysteines: a Tug of War Between Expression and Affinity


In a recombinant antibody scFv format, the presence of an unpaired cysteine (Cys) is implicated in reduced soluble expression and inefficient presentation in phage display. Compared to other species, antibodies derived from rabbits are more likely to contain this unpaired Cys residue at position 80 (Cys80), when generated in a scFv format. In a screening campaign to isolate rabbit scFv against cardiac troponin I (cTnI), it was found that, a large proportion of isolated cTnI-specific clones contained unpaired Cys80. To analyze the factors that led to the selection of anti-cTnI Cys80 scFv, after five rounds of biopanning, the biopanning experiments were repeated with a Cys80 scFv (MG4Cys), its alanine variant (MG4Ala), and an irrelevant high expressing scFv control. It was found that the selection and subsequent enrichment of MG4Cys scFv was ousted by the superior expressing variant MG4Ala, indicating that the Cys80 scFv was selected primarily due to its affinity. It is evident that phage-based selection is influenced by specific sequence characteristics affecting the expression as well as the binding specificity and this needs to be taken into account for selection of optimal antibody derivatives.

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The authors thank Prof. Carlos F. Barbas III (Scripps Research Institute) for kindly providing the pComb3 vector series used in this study.


All authors were supported by the Science Foundation Ireland (grants 05/CE3/B754 and 10/CE/B1821).

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Correspondence to B. Vijayalakshmi Ayyar.

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Ayyar, B.V., Hearty, S. & O’Kennedy, R. Decoding Selection Bias Imparted by Unpaired Cysteines: a Tug of War Between Expression and Affinity. Appl Biochem Biotechnol 185, 778–785 (2018).

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  • Rabbit antibodies
  • scFv
  • Unpaired cysteine
  • Phage display
  • Biopanning
  • Expression bias