Expression of TIMP-1 in Pichia pastoris. Selection of an anti-TIMP-1 specific single-chain Fv antibody from a large non-immune library
| Autor: | Florian Rüker, Mio Knezevic, Eva Obermayr, Matjaz Jeras, Gordana Wozniak |
|---|---|
| Rok vydání: | 2003 |
| Předmět: |
Phage display
medicine.drug_class Recombinant Fusion Proteins Clinical Biochemistry Enzyme-Linked Immunosorbent Assay Monoclonal antibody Biochemistry Pichia Pichia pastoris Antigen medicine Humans Cloning Molecular Panning (camera) Immunoglobulin Fragments Tissue Inhibitor of Metalloproteinase-1 biology Biochemistry (medical) Antibodies Monoclonal General Medicine Alkaline Phosphatase biology.organism_classification Fusion protein Molecular biology Recombinant Proteins biology.protein Antibody |
| Zdroj: | Clinica Chimica Acta. 327:171-179 |
| ISSN: | 0009-8981 |
| DOI: | 10.1016/s0009-8981(02)00372-8 |
| Popis: | To quantitate tissue inhibitor of metalloproteinase (TIMP)-1 in biological samples, a strategy for isolation of monoclonal antibodies was applied that employs a phage-displayed single-chain Fv (scFv). In order to obtain sufficient amounts of TIMP-1 to use as an antigen, high-level expression in Pichia pastoris was achieved under the control of the AOX-1 promotor. Purified protein antigen was then used for panning to achieve enrichment of specific phage from naive scFv library. In five subsequent panning rounds, antibody fragments that display specificity to TIMP-1 were selected. Regions encoding scFv were subcloned into a vector allowing production of scFv-alkaline phosphatase (AP) fusion proteins. Two such conjugates displaying dose-dependent reactivity with TIMP-1 were isolated and characterised, providing the basis for the construction of a TIMP-1 quantitation assay based entirely on recombinant proteins. |
| Databáze: | OpenAIRE |
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