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A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells
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An improved single-chain Fab platform for efficient display and recombinant expression.

James T Koerber1, Michael J Hornsby1, James A Wells1

  • 1Department of Pharmaceutical Chemistry, University of California, San Francisco, CA, 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA, 94158, USA; Recombinant Antibody Network, University of California, San Francisco, CA, 94158, USA.

Journal of Molecular Biology
|December 8, 2014
PubMed
Summary
This summary is machine-generated.

Researchers developed an improved single-chain fragment antigen binding (scFab) antibody format. This new scFab scaffold enhances display and bacterial expression, enabling high-yield production of stable, monomeric antibodies for research and therapeutics.

Keywords:
antibodybacterial and mammalian expressionhigh-throughput screeningphage displaysignal peptide

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Immunology

Background:

  • Antibody phage display is promising for generating recombinant antibodies.
  • Existing single-chain fragment antigen binding (scFab) formats face challenges with display levels and aggregation.
  • Optimized antibody scaffolds are crucial for advancing antibody generation platforms.

Purpose of the Study:

  • To develop an improved scFab format with enhanced display and bacterial expression.
  • To overcome limitations of previous scFab formats, including aggregation and low display.
  • To create a versatile scaffold for high-yield production of stable, monomeric antibodies.

Main Methods:

  • Engineered an scFab format retaining the interchain disulfide bond by increasing linker length.
  • Utilized co-translational signal recognition particle pathway and reengineered signal peptide for improved display.
  • Reformatted the scFab for expression in bacterial and mammalian hosts.

Main Results:

  • Achieved bacterial expression levels of 1-3 mg/L with the improved scFab format.
  • Obtained a 24-fold increase in display levels compared to the original scFab format.
  • Produced stable (Tm of 81 °C), predominantly monomeric (>90%) antibodies at high yields.

Conclusions:

  • The optimized scFab scaffold significantly improves antibody display and expression.
  • This new format facilitates high-throughput antibody generation for research and therapeutic applications.
  • The scFab format offers a stable, high-yield, and versatile platform for antibody discovery.