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

doi:10.3390/antib4020088

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 journal › Article › peer-review

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 language	English
Pages (from-to)	88-102
Number of pages	15
Journal	Antibodies
Volume	4
Issue number	2
DOIs	https://doi.org/10.3390/antib4020088
Publication status	Published - 15 May 2015

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

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10.3390/antib4020088

Codon-precise, synthetic, antibody fragment libraries built using automated hexamer codon additions
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.
Final published version, 1.09 MBLicence: CC BY 3.0

http://www.mdpi.com/2073-4468/4/2/88

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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. (2015). Codon-precise, synthetic, antibody fragment libraries built using automated hexamer codon additions and validated through next generation sequencing. Antibodies, 4(2), 88-102. https://doi.org/10.3390/antib4020088

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

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{\texttrademark}: 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.",

keywords = "colibra, proxiMAX randomization, saturation mutagenesis, CDR, single domain antibody, camelid antibody, antibody engineering",

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

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. ",

year = "2015",

month = may,

day = "15",

doi = "10.3390/antib4020088",

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journal = "Antibodies",

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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

TY - JOUR

T1 - Codon-precise, synthetic, antibody fragment libraries built using automated hexamer codon additions and validated through next generation sequencing

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.

N1 - 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.

PY - 2015/5/15

Y1 - 2015/5/15

N2 - 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.

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.

KW - colibra

KW - proxiMAX randomization

KW - saturation mutagenesis

KW - CDR

KW - single domain antibody

KW - camelid antibody

KW - antibody engineering

U2 - 10.3390/antib4020088

DO - 10.3390/antib4020088

M3 - Article

SN - 2073-4468

VL - 4

SP - 88

EP - 102

JO - Antibodies

JF - Antibodies

IS - 2

ER -

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

Abstract

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Keywords

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