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Expression of TIMP-1 in Pichia pastoris. Selection of an anti-TIMP-1 specific single-chain Fv antibody from a large non-immune library
Wozniak, Gordana ...
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. Purifiedprotein 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 displayingdose-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.
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