Cell-free expression of two single-chain monoclonal antibodies against lysozyme: effect of domain arrangement on the expression.

摘要: Single-chain antibodies (scFv), which can be produced in Escherichia coli cells, have been shown numerous cases to active antigen binding. In the case of two anti-lysozyme single-chain antibodies, scFvLH and scFvHL, reversed arrangement variable domains heavy light chains corresponding monoclonal expression level differs greatly when they are [Tsumoto et al. (1995) Biochem. Biophys. Res. Commun. 201, 546-551]. Although is high vivo, single chain antibody with orientation (scFvHL) was synthesized a very low yield no product could obtained. We report here synthesis these yields biological activities cell-free E. system presence reduced oxidized glutathione, protein disulfide isomerase (PDI), chaperones. immunological blotting assays, scFvs both arrangements exhibit specific binding activity antigens, hen egg-white lysozyme, an assay inhibited action lysozyme. mainly as expected molecular weight, whereas scFvHL additional shorter fragments, suggesting that isolation through vivo due mistranslation or ribonucleolytic cleavage transcript. scFv certain amount precipitated but chaperones soluble increased from 25 90% (PDI chaperones). The overall activity, however, were hardly influenced. reported provide significant amounts various fragments regardless order regions, including those expressed vivo.