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

Single-chain antibodies (scFv), which can be produced in Escherichia coil c ells, have been shown in numerous cases to be active in antigen binding. In the case of the two anti-lysozyme single-chain antibodies, scFv(LH) and sc Fv(HL)) which have the reversed arrangement of the variable domains of the heavy and light chains of the corresponding monoclonal antibodies, the expr ession level differs greatly when they are produced in Escherichia coil [Ts umoto et al, (1995) Biochem, Biophys, Res. Commun, 201, 546-551], Although the expression level of scFv(LH) 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. We report here the synthesis of t hese two anti-lysozyme single-chain antibodies in high yields and with high biological activities in a cell-free E. coil expression system in the pres ence of reduced and oxidized glutathione, protein disulfide isomerase (PDI) , and chaperones, In immunological blotting assays, the synthesized scFvs w ith both arrangements exhibit specific binding activity to the correspondin g antigens, hen egg-white lysozyme, and in an activity assay both inhibited the action of lysozyme. scFV(LH) is synthesized mainly as a product with t he expected molecular weight, whereas scFv(HL) is produced with additional shorter fragments, suggesting that the low yield isolation through the expr ession in vivo is due to mistranslation or ribonucleolytic cleavage of the transcript. In the cell-free expression of scFv a certain amount of the pro duct is precipitated but in the presence of chaperones the amount of solubl e protein increased from 25 to 90% (PDI and chaperones), The overall expres sion level and the specific biological activity, however, were hardly influ enced. 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.