Expression of TIMP-1 in Pichia pastoris. Selection of an anti-TIMP-1 specific single-chain Fv antibody from a large non-immune library.
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.
Wozniak
Gordana
G
Institute of Applied Microbiology, University of Agricultural Sciences, Muthgasse 18, 1190, Vienna, Austria.
Obermayr
Eva
E
Jeras
Matjaz
M
Knezevic
Mio
M
Rüker
Florian
F
D000469:Alkaline Phosphatase / Q000235:genetics; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D010843:Pichia / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000032:analysis* / Q000096:biosynthesis / Q000276:immunology*
D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D019715:Tissue Inhibitor of Metalloproteinase-1; C055966:immunoglobulin Fv; D000469:Alkaline Phosphatase