A Novel Dual Expression Platform for High Throughput Functional Screening of Phage Libraries in Product like Format

PLoS One. 2015 Oct 15;10(10):e0140691. doi: 10.1371/journal.pone.0140691. eCollection 2015.

Abstract

High throughput screenings of single chain Fv (scFv) antibody phage display libraries are currently done as soluble scFvs produced in E.coli. Due to endotoxin contaminations from bacterial cells these preparations cannot be reliably used in mammalian cell based assays. The monovalent nature and lack of Fc in soluble scFvs prevent functional assays that are dependent on target cross linking and/or Fc functions. A convenient approach is to convert scFvs into scFv.Fc fusion proteins and express them in mammalian cell lines for screening. This approach is low throughput and is only taken after primary screening of monovalent scFvs that are expressed in bacteria. There is no platform at present that combines the benefits of both bacterial and mammalian expression system for screening phage library output. We have, therefore, developed a novel dual expression vector, called pSplice, which can be used to express scFv.Fc fusion proteins both in E.coli and mammalian cell lines. The hallmark of the vector is an engineered intron which houses the bacterial promoter and signal peptide for expression and secretion of scFv.Fc in E.coli. When the vector is transfected into a mammalian cell line, the intron is efficiently spliced out resulting in a functional operon for expression and secretion of the scFv.Fc fusion protein into the culture medium. By applying basic knowledge of mammalian introns and splisosome, we designed this vector to enable screening of phage libraries in a product like format. Like IgG, the scFv.Fc fusion protein is bi-valent for the antigen and possesses Fc effector functions. Expression in E.coli maintains the speed of the bacterial expression platform and is used to triage clones based on binding and other assays that are not sensitive to endotoxin. Triaged clones are then expressed in a mammalian cell line without the need for any additional cloning steps. Conditioned media from the mammalian cell line containing the fusion proteins are then used for different types of cell based assays. Thus this system retains the speed of the current screening system for phage libraries and adds additional functionality to it.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bacteriophages / genetics
  • CHO Cells
  • Cloning, Molecular
  • Cricetinae
  • Cricetulus
  • HEK293 Cells
  • HL-60 Cells
  • High-Throughput Screening Assays / methods*
  • Humans
  • Immunoglobulin Fc Fragments / analysis
  • Immunoglobulin Fc Fragments / genetics
  • Peptide Library*
  • Single-Chain Antibodies / analysis*
  • Single-Chain Antibodies / isolation & purification

Substances

  • Immunoglobulin Fc Fragments
  • Peptide Library
  • Single-Chain Antibodies

Grants and funding

This study was funded solely by MedImmune which is a subsidiary of Astra-Zeneca Plc. MedImmune has provided support in the form of salaries for all the authors and procurement of all materials for the study, but did not have any additional role in the study design, data collection and analysis or preparation of the manuscript. It has approved the decision to publish. The specific roles of all the authors are articulated in the ‘author contributions’ section.