In vivo drafting of single‐chain antibodies for regulatory duty on the σ⁵⁴‐promoter Pu of the TOL plasmid

Abstract

The identification of single-chain antibodies (scFvs) that interfere in vivo with the building of the complex that activate the prokaryotic, sigma54-dependent promoter Pu of the catabolic TOL plasmid pWW0 is reported. To this end, a phage M13 library of scFvs was raised against the cognate prokaryotic enhancer-binding activator, XylR. The scFv pool was then expressed intracellularly in a reporter Pu-lacZ strain of Escherichia coli designed to permit formation of intramolecular disulphide bonds in cytoplasmic proteins. This strain allowed the assembly of functional scFvs and the direct testing of their activity on the Pu promoter in vivo. Specifically, genetic screening for lacZ-minus colonies yielded a number of scFvs able to downregulate transcriptional output in live cells. Two antibody clones were purified and shown to inhibit the activity of the same promoter in vitro as well. These scFvs targeted the DNA-binding domain of XylR and its ATP binding site respectively. This work provides a proof of principle that mimetic regulatory factors can be derived from an antibody repertoire that specifically interact with given transcriptional activators. As assembly of initiation complexes is stimulated or inhibited by regulatory proteins we argue that anti-XylR scFvs operate as bona fide transcriptional inhibitors of the Pu promoter.

Immunoglobulin domains inEscherichia coliand other enterobacteria: from pathogenesis to applications in antibody technologies

“…The high-stability properties of VHHs make them excellent candidates to function as intrabodies to abrogate or modulate the activity of intracellular proteins (Kirchhofer et al, 2010;Peréz-Martínez et al, 2010;Vercruysse et al, 2010). Using these strategies, immune libraries of scFvs have been screened in vivo for selection of intrabodies blocking a prokaryotic transcriptional activator (Jurado et al, 2006a) and a relaxase involved in plasmid conjugation in E. coli (Garcillan-Barcia et al, 2007). As most Ig V domains contain a conserved disulfide bond that is required for the stability of the folded polypeptide (Wörn & Plückthun, 2001), their expression in the reducing conditions found in the cytoplasm of E. coli mostly produce misfolded polypeptides unable to bind the antigen.…”

“…In this case, coexpression DssDsbC is not needed because Trx1 acts itself as a chaperone assisting the proper folding of the scFv (Jurado et al, 2006b). Using these strategies, immune libraries of scFvs have been screened in vivo for selection of intrabodies blocking a prokaryotic transcriptional activator (Jurado et al, 2006a) and a relaxase involved in plasmid conjugation in E. coli (Garcillan-Barcia et al, 2007). Intrabodies selected in vivo with E. coli trxB gor cells may be stabilized later for correct folding in the reducing environment of the cytoplasm of wild-type cells.…”

Monitoring biodegradative enzymes with nanobodies raised inCamelus dromedariuswith mixtures of catabolic proteins

“…Given the high diversity of the anti‐BphC V HH ‐M13 pool we reasoned that some molecular specimens of the antibodies could interact intracellularly with the protein surfaces of the dioxygenase in vivo , as this could make post‐translational control of its enzymatic activity feasible. To study this possibility we recloned in masse the whole of anti‐BphC V HH sequences from its original vector (pXylE5EHis, see Experimental procedures ) into the frame of the intracellular expression plasmid pTB7 (Jurado et al ., 2006). This vector adds a thioredoxin moiety to the N‐terminus of the V HH sequence, what is known to facilitate the stable folding of recombinant antibodies in an active form in the cytoplasm of bacteria (Jurado et al ., 2006).…”

“…To study this possibility we recloned in masse the whole of anti‐BphC V HH sequences from its original vector (pXylE5EHis, see Experimental procedures ) into the frame of the intracellular expression plasmid pTB7 (Jurado et al ., 2006). This vector adds a thioredoxin moiety to the N‐terminus of the V HH sequence, what is known to facilitate the stable folding of recombinant antibodies in an active form in the cytoplasm of bacteria (Jurado et al ., 2006). The pool of pBT7‐derived plasmids was then transformed into an E. coli strain carrying the bphC + plasmid pCKBphC.…”

“…For construction of pCKBphC (Cm R ), the PstI fragment from pDD5301 (Dowling et al ., 1993) containing bphC (encoding 2,3 OH biphenyl dioxygenase) was cloned into the low copy vector pCK01 (Fernández et al ., 1995). The pTC‐V HH (Ap R ) plasmid series harbouring V HH domain sequences expressed as protein fusions to thioredoxin were generated after purifying the corresponding NcoI/NotI fragments of the V HH pool first captured in vector pXylE5EHis (see above) and re‐cloning them into the same sites of the intracellular expression vector pTB7 (Jurado et al ., 2006).…”