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A High-Throughput Platform for the Generation of Synthetic Ab Clones by Single-Strand Site-Directed Mutagenesis

  • Eugenio GalloEmail author
Original paper
  • 31 Downloads

Abstract

Current developments in meta-data analysis and predictive computational models offer alternative routes for the identification of antibodies. In silico-based technologies and NGS data analysis from Ab phage-display selections offer expanded selections of Ab candidates. Accordingly, the identified de novo Abs with predicted selectivity for a target antigen must undergo rapid gene synthesis for downstream Ab characterizations. Here we describe a high-throughput strategy for the generation of synthetic Ab clones for expression as Fab proteins in Escherichia coli. Our approach utilizes simultaneous single-stranded site-directed mutagenesis of diversified Ab regions of a phagemid template with engineered complementary determining regions that contain multiple stop codon and restriction enzyme sites. Subsequently, we perform rapid screening of Ab DNA clones for correct gene assemblies by high-throughput Ab-phage protein expression screens. Identified sequences are corroborated by Sanger DNA sequencing analysis. In summary, our work describes a rapid and cost-effective platform for the high-throughput synthesis of synthetic Ab genes as Fab proteins for implementation into downstream protein validation pipelines.

Keywords

High-throughput Antibody Synthetic antibodies DNA gene synthesis Site-directed mutagenesis De novo 

Abbreviations

Abs

Antibodies

CDRs

Complementary determining regions

NGS

Next-generation sequencing

IPTG

Isopropyl β-d-1-thiogalactopyranoside

PBS

Phosphate-buffered saline

ssDNA

Single-stranded DNA

TMAC

Tetramethyl-ammonium-chloride

DTT

Dithiothreitol

BSA

Bovine serum albumin

HRP

Horseradish peroxidase

Notes

Acknowledgements

We would like to thank Y. Cabrera for optimization assistance with the ELISA detection screen and are also grateful to L. Moffat for helpful discussions and comments. This work was supported by the Charles H. Best Foundation.

Supplementary material

12033_2019_171_MOESM1_ESM.xlsx (17 kb)
Supplementary material 1 (XLSX 17 kb)
12033_2019_171_MOESM2_ESM.xlsx (16 kb)
Supplementary material 2 (XLSX 16 kb)

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Authors and Affiliations

  1. 1.Department of Molecular Genetics, Charles Best InstituteUniversity of TorontoTorontoCanada

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