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Protein Engineering vol. 4 no. 8 pp. 947-953, 1991
© 1991 Oxford University Press

RESEARCH-ARTICLE

Rapid detection of antigen binding by antibody fragments expressed in the periplasm of Escherichia coli

Jaymie R. Sawyer and Frederick R. Blattner

Department of Genetics, University of Wisconsin-Madison 445 Henry Mall, Madison, WI 53703, USA

Bacterial expression systems can greatly facilitate protein engineering of antibodies. We have developed a system for high-level expression of antibodies, antibody fragments, or hybrid antibodies with novel effector functions in the periplasm of Escherichia coli. From 5 ml of cells, a simple extraction yields sufficient material for SDS-gel electro-phoresis, detection and characterization of hapten binding. To demonstrate our system, heavy-chain variable regions and {lambda}1 light chains of a mouse anti-NP antibody were synthesized as hybrid proteins with a bacterial signal peptide (Omp F). Each chain is secreted into the periplasm where processing (cleavage of the signal peptide), folding and heterodimer association take place. Periplasmic proteins are released by cold osmotic shock, and hapten-binding activity is easily detected without further manipulation. The ease of genetic engineering in this system will facilitate the production of immunoglobulin derivatives designed for specific applications, and expression of these molecules in a native state will allow the rapid screening of combinatorial libraries and the results of mutagenesis.

Keywords: bacterial expression/immunoglobulin expression/protein engineering/recombinant antibodies/transport

Received April 6, 1991; revised September 19, 1991;
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