Cell-Free Expression of Two Single-Chain Monoclonal Antibodies against Lysozyme: Effect of Domain Arrangement on the Expression

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. We report here the synthesis 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 chaperones. 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.

Identification of factors impeding the production of a single-chain antibody fragment in Escherichia coli by comparing in vivo and in vitro expression

“…shown that certain scFvs can be expressed well in vitro but only in small quantities in vivo (Merk et al, 1999). By comparing the expression levels and ratios of active and inactive recombinant protein in both systems, it should be possible to identify some factors which limit the functional expression of scFvs in vivo.…”

Functional Production and Characterization of a Fibrin-Specific Single-Chain Antibody Fragment fromBacillus subtilis: Effects of Molecular Chaperones and a Wall-Bound Protease on Antibody Fragment Production

“…Although it is quite common to have V H at the N-terminal position in many SCA constructs, it has been reported in several cases that the presence of V H at the N-terminal position can severely affect both the production yield and biological activities of SCA fragments (2,4,7,45). Either mistranslation (32) or interference by the presence of a positively charged residue at the N terminus of V H (4) has been suggested to account for some of these observations. Therefore, the variable domains of MH-1 SCA were arranged in the order of V L -linker-V H .…”

Generation of hapten-specific recombinant antibodies: antibody phage display technology: a review

“…The two variable domains can be assembled into a single gene using a DNA linker either in the V H -linker-V L or V L -linker-V H form (Tsumoto et al, 1994;Ayala et al, 1995;Merk et al, 1999). Merk et al, 1999 presented that the expression level of scFv in N-termini-V L domain-linker-V H domain-C-termini orientation was relatively high but changing the order of variable domains causing a significant decrease in yield during in vitro expres-sion.…”

“…Merk et al, 1999 presented that the expression level of scFv in N-termini-V L domain-linker-V H domain-C-termini orientation was relatively high but changing the order of variable domains causing a significant decrease in yield during in vitro expres-sion. Therefore testing a suitable order of antibody domains is logical when considering the product yield.…”