| Budget Amount *help |
¥5,700,000 (Direct Cost: ¥5,700,000)
Fiscal Year 1998: ¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 1997: ¥3,200,000 (Direct Cost: ¥3,200,000)
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| Research Abstract |
Single-chain antibodies (scFv), which can be produced in Escherichia coli cells, have been shown in numerous cases to be active in antigen binding. In the case of the two anti-lysozyme single-chain antibodies, scFvLH and scFvHL, which have the reversed arrangement of the variable domains of the heavy and light chains of the corresponding monoclonal antibodies, the expression level differs greatly when they are produced in Escherichia coli [Tsumoto et al. (1995) Biochem. Biophys. Res. Commun. 201, 546-551]. Although the expression level of scFvLH is high in vivo, the single chain antibody with the reversed orientation (scFvHL) was synthesized in a very low yield and no active product could be obtained. In this study, we constructed the synthesis system of these two anti-lysozyme single-chain antibodies in high yields and with high biological activities in a cell-free E.coli expression system in the presence of reduced and oxidized glutathione, protein disulfide isomerase (PDI), and chap
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erones. In immunological blotting assays, the synthesized scFvs with both arrangements exhibit specific binding activity to the corresponding antigens, hen egg-white lysozyme, and in an activity assay both inhibited the action of lysozyme. scFvLH is synthesized mainly as a product with the expected molecular weight, whereas scFvHL is produced with additional shorter fragments, suggesting that the low yield isolation through the expression in vivo is due to mistranslation or ribonucleolytic cleavage of the transcript, In the cell-free expression of scFv a certain amount of the product is precipitated but in the presence of chaperones the amount of soluble protein increased from 25 to 90% (PDI and chaperones). The overall expression level and the specific biological activity, however, were hardly influenced. The system reported here can provide significant amounts of various scFv fragments regardless of the order of variable regions, including those which are hardly expressed in vivo. Based on the cell-free synthesis system, the expression of ribosomal protein 57 fused with HyHEL10 scFv LH was attempted, and the technical basis for molecular evolution of proteins has been constructed. Less
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