Codon-Precise, Synthetic, Antibody Fragment Libraries Built Using Automated Hexamer Codon Additions and Validated through Next Generation Sequencing

Antibodies

Laura Frigotto¹  Matthew E. Smith¹  Christopher Brankin¹  Ashni Sedani¹  Simon E. Cooper¹  Nisha Kanwar¹  Daniel Evans¹  Stanislava Svobodova¹  Claudia Baar¹  Jacob Glanville³  Christopher G. Ullman¹  Anna V. Hine² 

[1] Isogenica Ltd., The Mansion, Chesterford Research Park, Little Chesterford, Essex, CB10 1XL, UK; E-Mails:;School of Life and Health Sciences, Aston University, Aston Triangle, Birmingham, B4 7ET, UK;Distributed Bio Inc, 660 4th St, Suite 491, San Francisco, CA 94107, USA; E-Mail:

关键词: Colibra;    ProxiMAX randomization;    saturation mutagenesis;    CDR;    single domain antibody;    camelid antibody;    antibody engineering;   

DOI  :  10.3390/antib4020088

来源: mdpi

【 摘 要 】

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.

【 授权许可】

CC BY   
© 2015 by the authors; licensee MDPI, Basel, Switzerland.