| Author/Editor | Wozniak, Gordana; Obermayr, Eva; Jeras, Matjaž; Knežević, Mio; Rueker, Florian | |
| Title | Expression of TIMP-1 in Pichia pastoris. Selection of an anti-TIMP-1 specific single-chain Fv antibody from a large non-immune library | |
| Type | članek | |
| Source | Clin Chim Acta | |
| Vol. and No. | Letnik 327, št. 1-2 | |
| Publication year | 2003 | |
| Volume | str. 171-9 | |
| Language | eng | |
| Abstract | 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. | |
| Descriptors | METALLOPROTEINASES ALKALINE PHOSPHATASE RECOMBINANT FUSION PROTEINS PICHIA ESCHERICHIA COLI ENZYME-LINKED IMMUNOSORBENT ASSAY BACTERIOPHAGES SEQUENCE ANALYSIS, DNA POLYMERASE CHAIN REACTION CLONING, MOLECULAR BLOTTING, WESTERN ELECTROPHORESIS, POLYACRYLAMIDE GEL |