Enrichment of Escherichia coli spheroplasts displaying scFv antibodies specific for antigens expressed on the human cell surface

Anchored periplasmic expression (APEx) is a method for isolating high affinity ligand-binding proteins from large combinatorial libraries, and antibodies highly specific for soluble antigens were successfully isolated from APEx antibody libraries in combination with flow cytometric sorting (Harvey et al., Proc Natl Acad Sci USA 101(25):9193–9198, 2004). However, many disease markers and drug targets are localized on the cell surface, and often, unique posttranslational modifications and/or properly folded epitopes are lost when they were expressed and isolated in soluble form. In this study, we demonstrate that Escherichia coli spheroplasts, displaying antibodies and screened by a combination of plate-panning and flow cytometric sorting, can be used for isolating antibodies specific for antigens on the human cell surface. Two rounds of plate-panning followed by one round of flow cytometric sorting resulted in 7,200-fold enrichment of antibodies specific for the protective antigen of Bacillus anthracis from a large excess of spheroplasts expressing a scFv antibody to digoxin (a negative control). There is the potential to use this technique for library screening to find novel antibodies against disease cell surface antigens.     

Isolation of high-affinity ligand-binding proteins by periplasmic expression with cytometric screening (PECS)

Periplasmic expression with cytometric screening (PECS) is a powerful and rapid "display-less" technology for isolating ligand-binding proteins from diverse libraries. Escherichia coli expressing a library of proteins secreted into the periplasmic space are incubated with a fluorescent conjugate of the target ligand. Under the proper conditions, ligands as large as about 10 kDa can equilibrate within the periplasmic space without compromising the cell's integrity or viability. The bacterial cell envelope effectively serves as a dialysis bag to selectively retain receptor-fluorescent probe complexes but not free ligand. Cells displaying increased fluorescence are then isolated by flow cytometry. We demonstrate that scFv antibodies with both very high and low affinity to digoxigenin can be isolated from libraries screened by PECS using a benchtop flow cytometer. We also show that preexisting libraries constructed for display on filamentous bacteriophage can be screened by PECS without the need for subcloning. In fact, PECS was found to select for proteins that could be missed by conventional phage panning and screening methods.     

A periplasmic fluorescent reporter protein and its application in high-throughput membrane protein topology analysis

We have developed a periplasmic fluorescent reporter protein suitable for high-throughput membrane protein topology analysis in Escherichia coli. The reporter protein consists of a single chain (scFv) antibody fragment that binds to a fluorescent hapten conjugate with high affinity. Fusion of the scFv to membrane protein sites that are normally exposed in the periplasmic space tethers the scFv onto the inner membrane. Following permealization of the outer membrane to allow diffusion of the fluorescent hapten into the periplasm, binding to the anchored scFv renders the cells fluorescent. We show that cell fluorescence is an accurate and sensitive reporter of the location of residues within periplasmic loops. For topological analysis, a set of nested deletions in the membrane protein gene is employed to construct two libraries of gene fusions, one to the scFvand one to the cytoplasmic reporter green fluorescent protein (GFP). Fluorescent clones are isolated by flow cytometry and the sequence of the fusion junctions is determined to identify amino acid residues within periplasmic and cytoplasmic loops, respectively. We applied this methodology to the topology analysis of E. coli TatC protein for which previous studies had led to conflicting results. The ease of screening libraries of fusions by flow cytometry enabled the rapid identification of almost 90 highly fluorescent scFv and GFP fusions, which, in turn, allowed the fine mapping of TatC membrane topology.     

Anchoring proteins to Escherichia coli cell membranes using hydrophobic anchors derived from a Bacillus subtilis integral membrane protein

Anchored periplasmic expression (APEx) technology aims to express and localize proteins or peptides in the Escherichia coli periplasm. Some reports have suggested that transmembrane segments of integral membrane proteins can be used as membrane anchors in the APEx system. In this study, a series of hydrophobic anchors derived from the first putative transmembrane helix of a Bacillus subtilis integral membrane protein, MrpF, and its truncated forms were investigated for anchored periplasmic expression of alkaline phosphatase (PhoA) in E. coli. Anchoring efficiency of hydrophobic anchors was evaluated by monitoring the expression and activity of anchored PhoA. The length of hydrophobic anchors was found to be critical for anchoring proteins to cell membranes. This study may open new avenues for applying transmembrane segments derived from native membrane proteins as membrane anchors in the APEx system.     

Isolation of engineered, full-length antibodies from libraries expressed in Escherichia coli

We describe facile isolation of full-length IgG antibodies from combinatorial libraries expressed in E. coli. Full-length heavy and light chains are secreted into the periplasm, where they assemble into aglycosylated IgGs that are captured by an Fc-binding protein that is tethered to the inner membrane. After permeabilizing the outer membrane, spheroplast clones expressing so-called E-clonal antibodies, which specifically recognize fluorescently labeled antigen, are selected using flow cytometry. Screening of a library constructed from an immunized animal yielded several antibodies with nanomolar affinities toward the protective antigen of Bacillus anthracis.     

Surface display of antibodies

To screen antibody libraries that contain many millions of different clones, a selection system is required with an efficiency comparable to that of the immune system. This can be achieved by displaying antibodies on the surface of microorganisms containing the antibody's gene, analogous to the expression of the IgM antigen receptor on the surface of unactivated B-lymphocytes. Specific clones can then be selected using immobilized antigens. The minor coat protein of filamentous phages, pIII, which initiates the infection of E.coli by binding to their F-pili, and the major coat protein, pVIII, have been used as carriers for displaying antibodies on the phage surface. Recombinant antibodies have also been targeted to the cell surface of bacteria by fusing them with outer membrane components derived from lipoproteins, OmpA and an IgA protease. However, only the pIII system has been routinely used for screening antibody libraries. Here we describe the various antibody surface display systems and the screening of antibody libraries generated from the gene repertoire of lymphocytes and by gene synthesis. Finally, we have made a short comparison of the bacterial production of Fabs versus single chain antibodies (scFv).     

A human synthetic combinatorial library of arrayable single-chain antibodies based on shuffling in vivo formed CDRs into general framework regions

We describe a novel approach for high-throughput screening of recombinant antibodies, based on their immobilization on solid cellulose-based supports. We constructed a large human synthetic single-chain Fv antibody library where in vivo formed complementarity determining regions were shuffled combinatorially onto germline-derived human variable-region frameworks. The arraying of library-derived scFvs was facilitated by our unique display/expression system, where scFvs are expressed as fusion proteins with a cellulose-binding domain (CBD). Escherichia coli cells expressing library-derived scFv-CBDs are grown on a porous master filter on top of a second cellulose-based filter that captures the antibodies secreted by the bacteria. The cellulose filter is probed with labeled antigen allowing the identification of specific binders and the recovery of the original bacterial clones from the master filter. These filters may be simultaneously probed with a number of antigens allowing the isolation of a number of binding specificities and the validation of specificity of binders. We screened the library against a number of cancer-related peptides, proteins, and peptide-protein complexes and yielded antibody fragments exhibiting dissociation constants in the low nanomolar range. We expect our new antibody phage library to become a valuable source of antibodies to many different targets, and to play a vital role in facilitating high-throughput target discovery and validation in the area of functional cancer genomics.     

Raising antibodies against OprD, an outer membrane protein of Pseudomonas aeruginosa using translational fusions to MalE.

OprD is an outer membrane porin of Pseudomonas aeruginosa that mediates uptake of basic amino acids, peptides as well as carbapenem antibiotics. Polyclonal antibodies were raised against the OprD porin by creating protein fusions between the Escherichia coli maltose binding protein and four OprD fragments. These were expressed in E. coli and shown to be exported to the periplasm. The fusion proteins were purified by amylose affinity chromatography and used to immunize rabbits intramuscularly. We established that MalE fusions to OprD fragments retain maltose and amylose binding activities in vivo and in vitro, confirming proper folding of the MalE domain of hybrid proteins. Furthermore, we demonstrate that this strategy can be used to obtain specific antibodies against bacterial outer membrane proteins (OMPs).     

Characterization and localization of the KpsE protein of Escherichia coli K5, which is involved in polysaccharide export.

In Escherichia coli with group II capsules, the synthesis and cellular expression of capsular polysaccharide are encoded by the kps gene cluster. This gene cluster is composed of three regions. The central region 2 encodes proteins involved in polysaccharide synthesis, and the flanking regions 1 and 3 direct the translocation of the finished polysaccharide across the cytoplasmic membrane and its surface expression. The kps genes of the K5 polysaccharide, which is a group II capsular polysaccharide, have been cloned and sequenced. Region 1 contains the kpsE, -D, -U, -C, and -S genes. In this communication we describe the KpsE protein, the product of the kpsE gene. A truncated kpsE gene was fused with a truncated beta-galactosidase gene to generate a fusion protein containing the first 375 amino acids of beta-galactosidase and amino acids 67 to 382 of KpsE (KpsE'). This fusion protein was isolated and cleaved with factor Xa, and the purified KpsE' was used to immunize rabbits. Intact KpsE was extracted from the membranes of a KpsE-overexpressing recombinant strain with octyl-beta-glucoside. It was purified by affinity chromatography with immobilized anti-KpsE antibodies. Cytofluorometric analysis using the anti-KpsE antibodies with whole cells and spheroplasts, as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting (immunoblotting) of proteins from spheroplasts and membranes before and after treatment with proteinase K, indicated that the KpsE protein is associated with the cytoplasmic membrane and has an exposed periplasmic domain. By TnphoA mutagenesis and by constructing beta-lactamase fusions to the KpseE protein, it was possible to determine the topology of the KpsE protein within the cytoplasmic membrane.     

Improved expression characteristics of single-chain Fv fragments when fused downstream of the Escherichia coli maltose-binding protein or upstream of a single immunoglobulin-constant domain

The expression of single-chain Fv fragments (scFv) targeted to the periplasm of Escherichia coli often results in very low yields of soluble protein frequently accompanied by host cell growth arrest and sometimes lysis. Single-chain antibody fragments (scAb) are scFv with a human kappa light chain constant (HuCkappa) domain attached C-terminally and share similar problems of expression. By fusing the E. coli maltose-binding protein (mbp) gene either 3' or 5' to a scAb specific for the herbicide atrazine, a reduction in growth arrest was observed that was dependent on the order of gene fusion. The scAb-mbp fusion delayed the onset of growth arrest following induction while the mbp-scAb fusion appeared to ablate growth arrest completely. Cell fractionation revealed barely detectable levels of scAb-mbp in the periplasm while mbp-scAb was detected at equivalent levels as scAb in the periplasmic compartment, indicating that periplasmic scAb solubility is unrelated to propensity to cause growth arrest. IMAC purification of scAb and mbp-scAb proteins followed by liquid competition ELISA revealed the IC(50) for atrazine to be approximately 1 nM for both proteins demonstrating that 5'-mbp fusion does not alter antigen binding. The equivalent scFv and mbp-scFv vectors expressed far less material in both periplasmic and insoluble fractions indicating that the HuCkappa domain can have a positive effect on scFv expression when expressed either alone or as a mbp fusion. The ablation of growth arrest by a 5'-mbp fusion and enhancement of expression by a 3'-HuCkappa domain fusion were extended to a second scFv specific for the herbicide diuron. Therefore, by expressing scFv as tripartite fusions (mbp-scFv-HuCkappa) enhanced levels of soluble periplasmic expression can be achieved without causing growth arrest of the host cell, realizing the potential for constitutive expression of hapten-binding scFv in the E. coli periplasm.     

Engineering antibody fragments to fold in the absence of disulfide bonds

Disulfide bonds play a critical role in the stabilization of the immunoglobulin β-sandwich sandwich. Under reducing conditions, such as those that prevail in the cytoplasm, disulfide bonds do not normally form and as a result most antibodies expressed in that compartment (intrabodies) accumulate in a misfolded and inactive state. We have developed a simple method for the quantitative isolation of antibody fragments that retain full activity under reducing conditions from large mutant libraries. In E. coli, inactivation of the cysteine oxidoreductase DsbA abolishes protein oxidation in the periplasm, which leads to the accumulation of scFvs and other disulfide-containing proteins in a reduced form. Libraries of mutant scFvs were tethered onto the inner membrane of dsbA cells and mutants that could bind fluorescently labeled antigen in the reducing periplasm were screened by Anchored Periplasmic Expression (APEx; Harvey et al., Proc Natl Acad Sci USA 2004;101:9193–9198.). Using this approach, we isolated scFv antibody variants that are fully active when expressed in the cytoplasm or when the four Cys residues that normally form disulfides are substituted by Ser residues.     

Secretion and degradation of mutant leucine-specific binding protein molecules containing C-terminal deletions

The leucine-specific binding protein (LS-BP), a periplasmic component of the Escherichia coli high-affinity leucine transport system, is initially synthesized in a precursor form with a 23 amino acid N-terminal leader sequence that is removed during secretion of the protein into the periplasm. Using in vitro mutagenesis, deletion mutants of the LS-BP gene have been constructed with altered or missing amino acid sequences in the C-terminal portion of the protein. These altered binding proteins exhibited normal processing and secretion but were rapidly degraded in the periplasmic space. In the presence of an uncoupler of the transmembrane potential (CCCP) the precursor forms accumulated in the membrane and were protected from degradation. The altered binding proteins also were secreted by spheroplasts of E coli, after which they were easily detected.     

Use of phoA and lacZ fusions to study the membrane topology of ProW, a component of the osmoregulated ProU transport system of Escherichia coli.

The Escherichia coli ProU system is a member of the ATP-binding cassette (ABC) superfamily of transporters. ProU consists of three components (ProV, ProW, and ProX) and functions as a high-affinity, binding protein-dependent transport system for the osmoprotectants glycine betaine and proline betaine. The ProW protein is the integral inner membrane component of the ProU system. Its hydropathy profile predicts seven transmembrane spans and a hydrophilic amino terminus of approximately 100 residues, and it suggests the presence of an amphiphilic alpha-helix (L-61 to F-97) in close proximity to the first strongly hydrophobic segment of ProW. We have studied the membrane topology of the ProW protein by the phoA and lacZ gene fusion approach. A collection of 10 different proW-phoA fusions with alkaline phosphatase activity and 8 different proW-lacZ fusions with beta-galactosidase activity were isolated in vivo after TnphoAB and TnlacZ mutagenesis of a plasmid-encoded proW gene. The recovery of both enzymatically active ProW-PhoA and ProW-LacZ hybrid proteins indicates that segments of ProW are exposed on both sides of the cytoplasmic membrane. To compare the enzymatic activities of each of the indicator proteins joined at a particular site in ProW, we switched the phoA and lacZ reporter genes in vitro in each of the originally in vivo-isolated gene fusions. A mirror-like pattern in the enzyme activity of the resulting new ProW-PhoA and ProW-LacZ hybrid proteins emerged, thus providing positive signals for the location of both periplasmic and cytoplasmic domains in ProW. The protease kallikrein digests the amino-terminal tail of a ProW-LacZ hybrid protein in spheroplasts, suggesting that the amino terminus of ProW is located on the periplasmic side of the cytoplasmic membrane. From these data, a two-dimensional model for ProW was constructed; this model consists of seven transmembrane alpha-helices and an unusual amino-terminal tail of approximately 100 amino acid residues that protrudes into the periplasmic space.     

Matrix-assisted refolding of single-chain Fv- cellulose binding domain fusion proteins.

We describe a method for the isolation of recombinant single-chain antibodies in a biologically active form. The single-chain antibodies are fused to a cellulose binding domain as a single-chain protein that accumulates as insoluble inclusion bodies upon expression in Escherichia coli. The inclusion bodies are then solubilized and denatured by an appropriate chaotropic solvent, then reversibly immobilized onto a cellulose matrix via specific interaction of the matrix with the cellulose binding domain (CBD) moiety. The efficient immobilization that minimizes the contact between folding protein molecules, thus preventing their aggregation, is facilitated by the robustness of the Clostridium thermocellum CBD we use. This CBD is unique in retaining its specific cellulose binding capability when solubilized in up to 6 M urea, while the proteins fused to it are fully denatured. Refolding of the fusion proteins is induced by reducing with time the concentration of the denaturing solvent while in contact with the cellulose matrix. The refolded single-chain antibodies in their native state are then recovered by releasing them from the cellulose matrix in high yield of 60% or better, which is threefold or higher than the yield obtained by using published refolding protocols to recover the same scFvs. The described method should have general applicability for the production of many protein-CBD fusions in which the fusion partner is insoluble upon expression.     

A simple immunological detection method for the direct screening of genes from clone banks

A simple immunoassay has been developed which can be used in the isolation of particular gene(s) from a clone bank of recombinant plasmids. A clone bank of the DNA is constructed with a plasmid vector and maintained in Escherichia coli. The recombinant clones were filtered onto a hydrophobic grid membrane and grown up into individual colonies, and a replica was made onto nitrocellulose paper. The bacterial cells were then lysed with chloroform and the proteins were immobilized onto the nitrocellulose paper. The nitrocellulose paper is then reacted with a rabbit antibody preparation made against the particular antigenic product to detect the recombinant clone which carries the corresponding gene. The bound antibodies can be detected easily by a colorimetric assay using goat anti-rabbit antibodies conjugated to horseradish peroxidase. Positively reacting clones can be recovered from the master hydrophobic grid membrane filter for further characterization. We proposed to call this method "colony ELISA blot" and described the isolation of the genes coding for the soluble antigens of Pasteurella haemolytica using this method.     

Phage Peptide Libraries

Filamentous phage particles have been central in the construction of libraries displaying vast numbers of random peptides. These random peptides can be antigenically presented as fusions to coat proteins III and VIII of the phage. The isolation of ligate-reactive phage from an immense background of nonspecific phage is achieved by the biopanning process. Enrichment of reactive phage relative to unreactive phage occurs with alternate rounds of affinity selection to the desired molecular target and amplification of the specifically bound phage. This allows the isolation of rare binding species contained in the phage peptide libraries. Each phage particle contains the information in its genome pertaining to the type of random peptide insert displayed. Hence, the identification of binding motifs displayed on ligate-reactive phage is revealed by sequencing the relevant insert site in the phage genome. Phage peptide libraries have been used to isolate ligands to an array of protein ligates. The libraries have proved particularly effective in defining the binding sites of monoclonal antibodies and to some extent polyclonal sera. The analysis of the peptide insert sequences of a number of different clones of antibody binding phage can reveal a great deal about the nature and restriction of the amino acid residues critical for the antibody–antigen interaction.     

Mediation, by Saccharomyces cerevisiae translocation signals, of beta-lactamase transport through the Escherichia coli inner membrane and sensitive method for detection of signal sequences.

Signal sequences of Saccharomyces cerevisiae invertase and alpha-factor pheromone were tested for the ability to mediate protein transport through the inner membrane of Escherichia coli by fusion to bacterial beta-lactamase lacking the signal sequence (blaS0). Both types of transformants exhibited ampicillin resistance in accordance with the transport of the fused protein to the periplasmic compartment. This compartment contained most of the beta-lactamase activity present in the cell. Therefore, the tested yeast signal sequences, which conferred translocation of their proteins across the membrane of the endoplasmic reticulum in S. cerevisiae, can provide the same function in E. coli. The screening for ampicillin resistance among blaS0 fusions provides a convenient method for the isolation of functional yeast and possibly higher eucaryotic signal sequences.     

The use of gene fusions to examine the membrane topology of the L-subunit of the photosynthetic reaction center and of the cytochrome b subunit of the bc1 complex from Rhodobacter sphaeroides.

The topology of the cytochrome b subunit of the bc1 complex from Rhodobacter sphaeroides has been examined by generating gene fusions with alkaline phosphatase. Gene fusions were generated at random locations within the fbcB gene encoding the cytochrome b subunit. These fusion products were expressed in Escherichia coli and were screened for alkaline phosphatase activity on chromogenic plates. 33 in-frame fusions which showed activity were further characterized. The fusion junctions of all those fusions which had a high specific activity were clustered in three regions of the cytochrome b polypeptide, and thus these regions were tentatively assigned as being near the periplasmic surface. The data are consistent with a model containing eight transmembrane helices. In order to explore the validity of the gene fusion approach for a protein not normally expressed in E. coli, the topology of the L-subunit of the photosynthetic reaction center from R. sphaeroides was also explored using phoA gene fusions. A similar protocol was used as with the cytochrome b subunit. The gene fusions with high specific activity were shown to be in regions of the L-subunit polypeptide known to be at or near the periplasmic surface, as defined by the high resolution structure determined by X-ray crystallography. These data demonstrate the utility of this approach for determining membrane protein topology and extend potential applications to include at least some proteins not normally expressed in E. coli.     

Epitope distance to the target cell membrane and antigen size determine the potency of T cell-mediated lysis by BiTE antibodies specific for a large melanoma surface antigen

Melanoma chondroitin sulfate proteoglycan (MCSP; also called CSPG4, NG2, HMW-MAA, MSK16, MCSPG, MEL-CSPG, or gp240) is a surface antigen frequently expressed on human melanoma cells, which is involved in cell adhesion, invasion and spreading, angiogenesis, complement inhibition, and signaling. MCSP has therefore been frequently selected as target antigen for development of antibody- and vaccine-based therapeutic approaches. We have here used a large panel of monoclonal antibodies against human MCSP for generation of single-chain MCSP/CD3-bispecific antibodies of the BiTE (for bispecific T cell engager) class. Despite similar binding affinity to MCSP, respective BiTE antibodies greatly differed in their potency of redirected lysis of CHO cells stably transfected with full-length human MCSP, or with various MCSP deletion mutants and fusion proteins. BiTE antibodies binding to the membrane proximal domain D3 of MCSP were more potent than those binding to more distal domains. This epitope distance effect was corroborated with EpCAM/CD3-bispecific BiTE antibody MT110 by testing various fusion proteins between MCSP and EpCAM as surface antigens. CHO cells expressing small surface target antigens were generally better lysed than those expressing larger target antigens, indicating that antigen size was also an important determinant for the potency of BiTE antibody. The present study for the first time relates the positioning of binding domains and size of surface antigens to the potency of target cell lysis by BiTE-redirected cytotoxic T cells. In case of the MCSP antigen, this provides the basis for selection of a maximally potent BiTE antibody candidate for development of a novel melanoma therapy.