An efficient method for variable region assembly in the construction of scFv phage display libraries using independent strand amplification

Abstract

Phage display library technology is a common method to produce human antibodies. In this technique, the immunoglobulin variable regions are displayed in a bacteriophage in a way that each filamentous virus displays the product of a single antibody gene on its surface. From the collection of different phages, it is possible to isolate the virus that recognizes specific targets. The most common form in which to display antibody variable regions in the phage is the single chain variable fragment format (scFv), which requires assembly of the heavy and light immunoglobulin variable regions in a single gene.

In this work, we describe a simple and efficient method for the assembly of immunoglobulin heavy and light chain variable regions in a scFv format. This procedure involves a two-step reaction: (1) DNA amplification to produce the single strand form of the heavy or light chain gene required for the fusion; and (2) mixture of both single strand products followed by an assembly reaction to construct a complete scFv gene. Using this method, we produced 6-fold more scFv encoding DNA than the commonly used splicing by overlap extension PCR (SOE-PCR) approach. The scFv gene produced by this method also proved to be efficient in generating a diverse scFv phage display library. From this scFv library, we obtained phages that bound several non-related antigens, including recombinant proteins and rotavirus particles.

Keywords: :

• SOE-PCR
• independent strand amplification
• phage display library
• scFv assembly

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Dr. Jonas Chnaiderman for providing the Rotavirus particles, Karina Kramm for helping us in protein purification, and Nancy Fabres for her technical assistance. We also would like to thank Mr. Herve Camus for the technical support during ErbB3 diversity analysis. This study was supported by FONDEF-Chile D06I1005, Millennium Nucleus on Immunology and Immunotherapy (P07/088-F) and Millennium Institute on Immunology and Immunotherapy P09–016-F.