Specific Secretion of Active Single-Chain Fv Antibodies into the Supernatants ofEscherichia coliCultures by Use of the Hemolysin System

Abstract

A simple method for the nontoxic, specific, and efficient secretion of active single-chain Fv antibodies (scFvs) into the supernatants of Escherichia coli cultures is reported. The method is based on the well-characterized hemolysin transport system (Hly) of E. coli that specifically secretes the target protein from the bacterial cytoplasm into the extracellular medium without a periplasmic intermediate. The culture media that accumulate these Hly-secreted scFv's can be used in a variety of immunoassays without purification. In addition, these culture supernatants are stable over long periods of time and can be handled basically as immune sera.The current methodology for the selection and production of antibody fragments in Escherichia coli involves the generation of large repertories of human or murine immunoglobulin (Ig) V gene segments, either from naive or immunized individuals, and its cloning in filamentous phage (or phagemid) vectors that allow both the phage display and the production of the reconstructed antibody Fv fragments (17,19,25,27). After a selection (biopanning) of Fv clones capable of binding a given antigen, the recombinant Fv antibodies are produced individually in E. coli and tested for their antigen-binding properties (16,22). The standard Ig fragments produced in E. coli are the so-called single-chain Fv (scFv) in which the variable domains from the heavy (V H ) and light (V L ) chains are linked in a single polypeptide. The standard protocol for production of scFv's require their translocation to the periplasmic space using an N-terminal signal peptide (SP) that is recognized by the general secretion pathway of E. coli, encoded by the sec genes, and which is responsible for the export of most cellular proteins targeted to the extracytoplasmic compartments (12, 31). Next, the scFv polypeptides are purified, using chromatographic techniques, from periplasmic protein extracts obtained from those cells (30).Besides being time-consuming, the major problem associated with the production of scFv in E. coli is the toxicity caused by their periplasmic export and accumulation, which eventually leads to the lysis of the bacterial cell (25, 30). The export of scFvs gives rise to a number of toxic effects, such as the jamming of the Sec pathway, the titration of periplasmic-folding catalysts, the induction of periplasmic proteases, and an enhanced outer membrane permeability (3,6,7,20,32). All of these events have important biotechnological consequences, such as low production yields and the formation of scFv aggregates. Thus, an ideal method for scFv production should allow their secretion to the extracellular space without a periplasmic intermediate and by a Sec-independent pathway.The hemolysin transport system (Hly) is a type I secretory apparatus that forms a protein channel between the inner and outer membranes of E. coli through which the hemolysin toxin (HlyA) is secreted (5). The protein machinery of Hly is independent of the cellular sec genes and consists in two inner membrane components, ...

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“…It may be that fusion to the 30-kDa HlyA signal abolished function of the scFv or caused misfolding of the scFv. It may also be that secretion via the periplasm (where essential disulfide bonds in the structure of an scFv can be formed) is a requirement for activity, although there is one report in the literature of secretion of a functional scFv fused to the HlyA C terminus (13).…”

“…Replacing the bla gene in pEHLYA2-SD with aac(3)IV resulted in the hlyA secretion construct pDB47 (Fig. 1B) (13). This was done using recombineering and a conjugation as described for pDB36.…”

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“…The protein machinery of the Hly type I secretory apparatus consists of two operon-specific inner membrane components, HlyB and HlyD, and the chromosomally encoded outer membrane protein, TolC, which form a protein channel between the inner and outer membranes. The HlyB-HlyD complex recognizes the C-terminal portion of the Hly enzyme (HlyA), thereby allowing the secretion of polypeptides fused to this signal sequence (8,9).…”

“…To construct the C 52 -HlyA 218 (Hly, hemolysin) expression plasmid, the region of HIV NL4.3 -encoding amino acids 114-162 of gp41 was cloned into pEHLYA2-SD, a high-copy-number ampicillin resistance (Ap r ) plasmid that contains the lac operon promoter, a Shine-Dalgarno sequence, multiple cloning sites, and an epitope tag (Etag) in frame with the C-terminal 218 aa of HlyA (8). The recombinant plasmid was contransformed with the low copy number chloramphenicol resistance (Cm r ) plasmid pVDL9.3 (9), which provides the HlyB and HlyD transporters in trans, into Escherichia coli Nissle 1917 (obtained from a commercial preparation of the probiotic Mutaflor from Ardeypharm, Herdecke, Germany).…”

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“…Expression of scFv in transgenic plants has been proposed as a way to produce and store pharmaceutical antibodies (Stoger et al, 2000 ;Yuan et al, 2000) and as a means to block physiological factors or plant pathogens (Le Gall et al, 1998 ;Conrad & Fiedler, 1998). Most recombinant scFv molecules have been expressed in Escherichia coli cells, but results were variable whether or not the scFv was fused with a reporter protein (Fernandez et al, 2000 ;Fernandez & De Lorenzo, 2001). ScFv accumu- lation as insoluble inclusion bodies, needing solubilization and refolding, has been reported to be a frequent problem (Smallshaw et al, 1999 ;Sanchez et al, 1999 ;Cho et al, 2000), triggering the search for alternative hosts.…”