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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.

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G protein-coupled receptors, like many other membrane proteins, are notoriously difficult to synthesize in conventional cellular systems. Although expression in insect cells is considered the preferred technique for structural characterizations in particular, inefficient membrane translocation, instability, toxic effects and low yields still pose clear limitations for their production in living cells. Recent studies started to explore alternative strategies for the in vitro production of problematic membrane proteins in cell-free lysates in combination with supplied membranes. We provide a detailed study on the production efficiencies and quality of G protein-coupled receptors, Fab fragments and other proteins synthesized in insect cell lysates containing endogenous microsomes. Effects of different reaction kinetics, redox conditions and sample preparations on the specific activities of synthesized proteins have been analyzed. The extent of glycosylation, membrane translocation and percentages of ligand binding active fractions of synthesized protein samples have been determined. We provide strong evidence that membrane insertion of integral membrane proteins can represent a prime limiting factor for their preparative scale in vitro production. Improved expression protocols resulting into higher production rates yielded more active protein in case of Fab fragments, but not in case of the human endothelin B receptor.

A eukaryotic cell-free system based on Spodoptera frugiperda cells was developed for the convenient synthesis of Fab antibody fragments and other disulfide bridge containing proteins. The system uses (i) a cell lysate that is mildly prepared under slightly reduced conditions, thus maintaining the activity of vesicles derived from the endoplasmic reticulum, (ii) signal peptide dependent translocation into these vesicles, and (iii) a redox potential based on reduced and oxidized glutathione. Monomeric heavy and light immunoglobulin chains are almost completely converted to highly active dimeric Fab joined by intermolecular disulfide bridges without supplementation of chaperones or protein disulfide isomerase. The applicability of the system is demonstrated by the synthesis of anti-lysozyme and anti-CD4 Fab antibody fragments yielding approximately 10 g Fab per milliliter reaction mixture. The lack of endotoxins in this system is a prerequisite that synthesized Fab can be applied directly using whole synthesis reactions in cell-based assays that are sensitive to this substance class. Moreover, the system is compatible with PCR-generated linear templates enabling automated generation of antibody fragments in a high-throughput manner, and facilitating its application for screening and validation purposes.

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Cell-free protein synthesis (CFPS) is a fast and convenient way to synthesize proteins for analytical studies and applications. CFPS, when equipped with a suitable orthogonal pair, allows for protein-site-directed labeling with desired functionalities such as fluorescent dyes or therapeutic groups that are needed to tailor proteins for analytical applications. In this context, chemoselective reactive pyrrolysine analogues (CR-OAs) are of particular value, as this class of unnatural amino acids, among other useful properties, covers a wide range of different chemoselective reactions. In this study, we present a flexible approach that facilitates incorporation of CR-OAs in CFPS systems. In particular, a fairly simple addition of two expression plasmids in our cell-free system, one encoding pyrrolysyl-tRNA synthetase and the other one the target protein, enabled ribosomal synthesis of proteins in the half-milligram range with the pre-installed orthogonal reactivity, easily modifiable by using mild, copper-free bioorthogonal chemistry. Our CFPS system allows rapid and highly customizable expression, as shown by several examples of successful site-directed fluorescence labeling. The feasibility of our CFPS system for protein analytics is further proved by demonstrating the functional integrity of a labeled protein by interaction measurements using microscale thermophoresis.