Use of antibody gene library for the isolation of specific single chain antibodies. by ampicillin-antigen conjugates
| Autor: | Katrin Messerschmidt, Nicole Grenz, Katja Heilmann, Meina Neumann-Schaal |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2013 |
| Předmět: |
Phage display
biology Immunology Genetic Vectors Antibody Affinity Gene Expression Fusion protein Molecular biology Antibodies Mice Antigen Ribosome display Gene Order biology.protein Immunology and Allergy Animals Genomic library Ampicillin Antibody Antigens Direct fluorescent antibody Single-Chain Antibodies Institut für Biochemie und Biologie Gene Library |
| Popis: | Isolation of recombinant antibodies from antibody libraries is commonly performed by different molecular display formats including phage display and ribosome display or different cell-surface display formats. We describe a new method which allows the selection of Escherichia coli cells producing the required single chain antibody by cultivation in presence of ampicillin conjugated to the antigen of interest. The method utilizes the neutralization of the conjugate by the produced single chain antibody which is secreted to the periplasm. Therefore, a new expression system based on the pET26b vector was designed and a library was constructed. The method was successfully established first for the selection of E. coli BL21 Star (DE3) cells expressing a model single chain antibody (anti-fluorescein) by a simple selection assay on LB-agar plates. Using this selection assay, we could identify a new single chain antibody binding biotin by growing E. coli BL21 Star (DE3) containing the library in presence of a biotin–ampicillin conjugate. In contrast to methods as molecular or cell surface display our selection system applies the soluble single chain antibody molecule and thereby avoids undesired effects, e.g. by the phage particle or the yeast fusion protein. By selecting directly in an expression strain, production and characterization of the selected single chain antibody is possible without any further cloning or transformation steps. |
| Databáze: | OpenAIRE |
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