Codon-Precise, Synthetic, Antibody Fragment Libraries Built Using Automated Hexamer Codon Additions and Validated through Next Generation Sequencing
by
Laura Frigotto 1, Matthew E. Smith 1, Christopher Brankin 1,†, Ashni Sedani 1, Simon E. Cooper 1, Nisha Kanwar 1, Daniel Evans 1, Stanislava Svobodova 1,‡, Claudia Baar 1, Jacob Glanville 2, Christopher G. Ullman 1,* and Anna V. Hine 3,*
Laura Frigotto 1, Matthew E. Smith 1, Christopher Brankin 1,†, Ashni Sedani 1, Simon E. Cooper 1, Nisha Kanwar 1, Daniel Evans 1, Stanislava Svobodova 1,‡, Claudia Baar 1, Jacob Glanville 2, Christopher G. Ullman 1,* and Anna V. Hine 3,*
1
Isogenica Ltd., The Mansion, Chesterford Research Park, Little Chesterford, Essex, CB10 1XL, UK
2
Distributed Bio Inc, 660 4th St, Suite 491, San Francisco, CA 94107, USA
3
School of Life and Health Sciences, Aston University, Aston Triangle, Birmingham, B4 7ET, UK
*
Authors to whom correspondence should be addressed.
†
Current address: MedImmune, Milstein Building, Granta Park, Cambridge, CB21 6GH, UK.
‡
Current address: Healthcare Diagnostics Ltd., Rossington Place, Graphite Way, Hadfield, Glossop, Derbyshire SK13 1QG, UK.
Academic Editor: Dimiter S. Dimitrov
Antibodies 2015, 4(2), 88-102; https://doi.org/10.3390/antib4020088
Received: 5 March 2015 / Revised: 21 April 2015 / Accepted: 11 May 2015 / Published: 15 May 2015
(This article belongs to the Special Issue Antibody Engineering)
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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Keywords:
Colibra; ProxiMAX randomization; saturation mutagenesis; CDR; single domain antibody; camelid antibody; antibody engineering
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MDPI and ACS Style
Frigotto, L.; Smith, M.E.; Brankin, C.; Sedani, A.; Cooper, S.E.; Kanwar, N.; Evans, D.; Svobodova, S.; Baar, C.; Glanville, J.; Ullman, C.G.; Hine, A.V. Codon-Precise, Synthetic, Antibody Fragment Libraries Built Using Automated Hexamer Codon Additions and Validated through Next Generation Sequencing. Antibodies 2015, 4, 88-102.
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