BIOL 31 |
| c-Met, a high affinity receptor for hepatocyte growth factor/scatter factor, shown to be overexpressed in a variety of malignant cells, is a potential biomarker as well as a therapeutic target. Thus antibody specific for c-Met is expected to be efficiently employed in the clinical treatment or imaging of many cancer cells. In our previous study, we have constructed the scFv against c-Met and could functionally express the scFv in the cytoplasm of E. coli by using trx, gor deleted mutant as a host and by coexpressing chaperones. However, when the scFv against c-Met was cloned and expressed in the cytoplasm of E. coli, the expression level of the protein was much lower than expected. Here we show that the domain order of VL and VH can significantly affect the expression levels of scFvs and diabodies against c-Met. ScFvs and diabodies showing two different domain orders, i.e. VL-linker-VH and VH-linker-VL, were expressed under T7 promoter and their activities as well as their productivities were compared. In addition, expression conditions to achieve a functional and soluble expression of the fragment antibodies were investigated. To purify the anti-c-Met scFv simply, we fused hexahistidine residues at the C-terminus of the fragment antibodies and purified by immobilized metal affinity chromatography (IMAC). Soluble scFvs and diabodies produced in this manner ware characterized for their antigen-binding characteristics. |
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Protein Structure and Folding
4:30 PM-6:30 PM, Sunday, 10 September 2006 Moscone Center -- Hall D, Poster
Division of Biological Chemistry |