Codon-precise, synthetic, antibody fragment libraries built using automated hexamer codon additions and validated through next generation sequencing

Laura Frigotto, Matthew E. Smith, Christopher Brankin, Ashni Sedani, Simon E. Cooper, Nisha Kanwar, Daniel Evans, Stanislava Svobodova, Claudia Baar, Jacob Glanville, Christopher G. Ullman, Anna V. Hine

Research output: Contribution to journalArticle

Abstract

We have previously described ProxiMAX, a technology that enables the fabrication of precise, combinatorial gene libraries via codon-by-codon saturation mutagenesis. ProxiMAX was originally performed using manual, enzymatic transfer of codons via blunt-end ligation. Here we present Colibra™: an automated, proprietary version of ProxiMAX used specifically for antibody library generation, in which double-codon hexamers are transferred during the saturation cycling process. The reduction in process complexity, resulting library quality and an unprecedented saturation of up to 24 contiguous codons are described. Utility of the method is demonstrated via fabrication of complementarity determining regions (CDR) in antibody fragment libraries and next generation sequencing (NGS) analysis of their quality and diversity.
Original languageEnglish
Pages (from-to)88-102
Number of pages15
JournalAntibodies
Volume4
Issue number2
DOIs
Publication statusPublished - 15 May 2015

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Immunoglobulin Fragments
Codon
Complementarity Determining Regions
Gene Library
Mutagenesis
Libraries
Ligation
Technology
Antibodies

Bibliographical note

This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Keywords

  • colibra
  • proxiMAX randomization
  • saturation mutagenesis
  • CDR
  • single domain antibody
  • camelid antibody
  • antibody engineering

Cite this

Frigotto, Laura ; Smith, Matthew E. ; Brankin, Christopher ; Sedani, Ashni ; Cooper, Simon E. ; Kanwar, Nisha ; Evans, Daniel ; Svobodova, Stanislava ; Baar, Claudia ; Glanville, Jacob ; Ullman, Christopher G. ; Hine, Anna V. / Codon-precise, synthetic, antibody fragment libraries built using automated hexamer codon additions and validated through next generation sequencing. In: Antibodies. 2015 ; Vol. 4, No. 2. pp. 88-102.
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abstract = "We have previously described ProxiMAX, a technology that enables the fabrication of precise, combinatorial gene libraries via codon-by-codon saturation mutagenesis. ProxiMAX was originally performed using manual, enzymatic transfer of codons via blunt-end ligation. Here we present Colibra™: an automated, proprietary version of ProxiMAX used specifically for antibody library generation, in which double-codon hexamers are transferred during the saturation cycling process. The reduction in process complexity, resulting library quality and an unprecedented saturation of up to 24 contiguous codons are described. Utility of the method is demonstrated via fabrication of complementarity determining regions (CDR) in antibody fragment libraries and next generation sequencing (NGS) analysis of their quality and diversity.",
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Frigotto, L, Smith, ME, Brankin, C, Sedani, A, Cooper, SE, Kanwar, N, Evans, D, Svobodova, S, Baar, C, Glanville, J, Ullman, CG & Hine, AV 2015, 'Codon-precise, synthetic, antibody fragment libraries built using automated hexamer codon additions and validated through next generation sequencing', Antibodies, vol. 4, no. 2, pp. 88-102. https://doi.org/10.3390/antib4020088

Codon-precise, synthetic, antibody fragment libraries built using automated hexamer codon additions and validated through next generation sequencing. / Frigotto, Laura; Smith, Matthew E.; Brankin, Christopher; Sedani, Ashni; Cooper, Simon E.; Kanwar, Nisha; Evans, Daniel; Svobodova, Stanislava; Baar, Claudia; Glanville, Jacob; Ullman, Christopher G.; Hine, Anna V.

In: Antibodies, Vol. 4, No. 2, 15.05.2015, p. 88-102.

Research output: Contribution to journalArticle

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AU - Frigotto, Laura

AU - Smith, Matthew E.

AU - Brankin, Christopher

AU - Sedani, Ashni

AU - Cooper, Simon E.

AU - Kanwar, Nisha

AU - Evans, Daniel

AU - Svobodova, Stanislava

AU - Baar, Claudia

AU - Glanville, Jacob

AU - Ullman, Christopher G.

AU - Hine, Anna V.

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Y1 - 2015/5/15

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AB - We have previously described ProxiMAX, a technology that enables the fabrication of precise, combinatorial gene libraries via codon-by-codon saturation mutagenesis. ProxiMAX was originally performed using manual, enzymatic transfer of codons via blunt-end ligation. Here we present Colibra™: an automated, proprietary version of ProxiMAX used specifically for antibody library generation, in which double-codon hexamers are transferred during the saturation cycling process. The reduction in process complexity, resulting library quality and an unprecedented saturation of up to 24 contiguous codons are described. Utility of the method is demonstrated via fabrication of complementarity determining regions (CDR) in antibody fragment libraries and next generation sequencing (NGS) analysis of their quality and diversity.

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KW - proxiMAX randomization

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