Rapid detection of antigen binding by antibody fragments expressed in the periplasm of Escherichia coli.
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 electrophoresis, 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.
Sawyer
J R
JR
Department of Genetics, University of Wisconsin-Madison 53703.
Blattner
F R
FR
D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D001666:Binding Sites, Antibody; D002462:Cell Membrane / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005803:Genes, Immunoglobulin / Q000235:genetics*; D006241:Haptens / Q000276:immunology; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D051379:Mice; D009596:Nitrophenols / Q000276:immunology; D010648:Phenylacetates; D015202:Protein Engineering / Q000379:methods*; D021382:Protein Sorting Signals / Q000235:genetics
D001425:Bacterial Outer Membrane Proteins; D006241:Haptens; D007128:Immunoglobulin Fragments; D009596:Nitrophenols; D010648:Phenylacetates; D021382:Protein Sorting Signals; C026242:4-hydroxy-5-nitrophenyl acetic acid