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Functional expression of a single-chain anti-progesterone antibody fragment in the cytoplasm of a mutant Escherichia coli.

Expression of antibody fragments (Fab, Fv, single chain sFv) in Escherichia coli is widely used in antibody engineering (1). Antibodies contain a number of intrachain disulphide bonds which are essential for correct folding and hence antigen binding. To facilitate disulphide bond formation, antibody fragments have been expressed and secreted into E.coli periplasm, an oxidising milieu, and shown to be functionally and structurally identical to the native antibody (1-3). In contrast, antibody fragments expressed in the cytoplasm of E.coli formed inactive inclusion bodies, requiring denaturation and refolding in vitro to recover binding activity (1). Here, we report a strategy for cytoplasmic production of a functional single-chain antibody fragment in an E.coli mutant strain AD494, in which disulphide bond formation occurs in the cytoplasm due to mutation in thioredoxin reductase (4). This host has been reported to produce functional alkaline phosphatase and mouse urokinase, both of which require disulphide bridges, in the cytoplasm (4). The advantages for production of antibody fragments are a potentially higher yield from the cytoplasm than the periplasm and avoidance of the problem of protein sequences incompatible with E.coli translocation machinery (5). We have previously described functional expression in E.coli periplasm of a DNA construct encoding a three-domain singlechain fragment of an anti-progesterone antibody, DB3 (6). In this fragment, designated VH/K, the VH domain is linked to the complete K chain, via the VH-CH1 elbow sequence. Its functional characteristics of affinity, specificity and idiotypy resemble closely those of the native DB3 antibody (6). To test the possibility of producing this fragment in E.coli cytoplasm, we have constructed an expression plasmid (pUVH/K) by ligating DNA encoding VH/K into plasmid pUC19. E.coli host AD494 containing pUVH/K was grown overnight at 37°C in 2 ml 2x TY medium containing 100 .g/ml ampicillin. After transferring to fresh 100 ml 2x TY medium (100 jig/ml ampicillin), growth was continued at 37°C to OD6Mo 0.6-0.8. IPTG to 2 mM final concentration was added, and induction carried out at 25°C for 4 h. After harvesting by centrifugation, the cell pellet was resuspended in 10 ml buffer containing 50 mM glucose, 25 mM Tris-HCl pH 8.0, 10 mM EDTA and 2 mg/ml lysozyme. The mixture was incubated on ice for 30 min, sonicated three times for 30 s, and centrifuged at 11 000 g at 4°C for 10 min; the supernatant (cytoplasmic extract) was used to examine binding activity. Periplasmic extracts containing VH/K were produced in E.coli D29A1 transformed by pCombVH/K, as described (6). Quantitation of soluble extracted material was by SDS-PAGE, Western blotting using horseradish peroxidase-coupled antimouse K light chain as developing reagent and densitometry (6), and functionality of the VH/K fragments was tested by binding to progesterone-coupled BSA in ELISA (6). In both, known amounts of progesterone-binding DB3 Fab were used as standard to detenmine yields. When tested for progesterone binding activity, VH/K from cytoplasmic extracts of AD494 exhibited very similar affmiity, specificity and idiotypic profiles to VH/K produced in the periplasm of D29A1. Figure 1 shows a specificity analysis comparing VH/K from cytoplasmic and periplasmic extracts, demonstrating that they both have equivalent binding of progesterone, cross-react with aetiocholanolone with the same relative affinities, and fail to recognise testosterone. As determined by ELISA, binding activity of soluble functional material in AD494 cytoplasm was about five times higher than that in periplasmic extracts from identical numbers of E.coli D29A1 (Fig. 2). Similarly prepared supernatant from control cells (E.coli AD494 containing pUC 19) had no binding affinity for progesterone (Fig. 2). SDS-PAGE and Western blotting analysis of total E.coli AD494 containing PUVH/K showed that slightly slower migration of VH/K occurred after reduction with dithiothreitol, compared with non-reducing conditions, indicating the formation of intrachain disulphide bonds in the cytoplasm (not shown). No dimeric form of VH/K was detected, despite the C-terminal cysteine. Quantitative yields ofVH/K were determined in the cytoplasm and periplasm of the E.coli hosts AD494, D29A1 and XL1-blue, using Western blotting for protein quantitation and ELISA to compare funtional binding activities. The soluble protein yields and binding activities were as follows: 500 ,ug/l each in AD494 cytoplasm and periplasm, both 100% active; 875 jg/l and 125 jg/l in D29A1 cytoplasm and periplasm, with 10 and 100% activity respectively; and 1.2 mg/l and 35 jg/I in XLI-blue cytoplasm and periplasm, also with 10 and 100% respective activities. These results demonstrate the relatively low functional