Joachim Jose

The Autodisplay Story, from Discovery to Biotechnical and Biomedical Applications

SUMMARY Among the pathways used by gram-negative bacteria for protein secretion, the autotransporter pathway represents a solution of impressive simplicity. Proteins are transported, independent of their nature as recombinant or native passengers, as long as the coding nucleotide sequence is inserted in frame between those of an N-terminal signal peptide and a C-terminal domain, referred to as the β-barrel of the outer membrane translocation unit. The immunoglobulin A1 (IgA1) protease from Neisseria gonorrhoeae was the first identified member of the autotransporter family of secreted proteins. The IgA1 protease was employed in initial experiments investigating autotransporter-mediated surface display of recombinant proteins and to investigate structural and functional requirements. Various other autotransporter proteins have since been described, and the autodisplay system was developed on the basis of the natural Escherichia coli autotransporter protein AIDA-I (adhesin involved in diffuse adherence). Autodisplay has been used for the surface display of random peptide libraries to successfully screen for novel enzyme inhibitors. The autodisplay system was also used for the surface display of functional enzymes, including esterases, oxidoreductases, and electron transfer proteins. Whole E. coli cells displaying enzymes have been utilized to efficiently synthesize industrially important rare organic compounds with specific chirality. Autodisplay of epitopes on the surface of attenuated Salmonella carriers has also provided a novel way to induce immune protection after oral vaccination. This review summarizes the structural and functional features of the autodisplay system, illustrating its discovery and most recent applications. Autodisplay facilitates the export of more than 100,000 recombinant molecules per single cell and permits the oligomerization of subunits on the cell surface as well as the incorporation of inorganic prosthetic groups after transport of apoproteins onto the bacterial surface without disturbing bacterial integrity or viability. We discuss future biotechnical and biomedical applications in the light of these achievements.

Autodisplay: efficient bacterial surface display of recombinant proteins

To display a protein or peptide with a distinct function at the surface of a living bacterial cell is a challenging exercise with constantly increasing impact in many areas of biochemistry and biotechnology. Among other systems in Gram-negative bacteria, the Autodisplay system provides striking advantages when used to express a recombinant protein at the surface of Escherichia coli or related bacteria. The Autodisplay system has been developed on the basis of and by exploiting the natural secretion mechanism of the AIDA-I autotransporter protein. It offers the expression of more than 105 recombinant molecules per single cell, permits the multimerization of subunits expressed from monomeric genes at the cell surface, and allows, after transport of an apoprotein to the cell surface, the incorporation of an inorganic prosthetic group without disturbing cell integrity or cell viability. Moreover, whole cells displaying recombinant proteins by Autodisplay can be subjected to high-throughput screening (HTS) methods such as ELISA or FACS, thus enabling the screening of surface display libraries and providing access to directed evolution of the recombinant protein displayed at the cell surface. In this review, the application of the Autodisplay system for the surface display of enzymes, enzyme inhibitors, epitopes, antigens, protein and peptide libraries is summarised and the perspectives of the system are discussed.

Absence of periplasmic DsbA oxidoreductase facilitates export of cysteine-containing passenger proteins to the Escherichia coli cell surface via the Igaβ autotransporter pathway

The Iga beta autotransporter function of IgA1 protease from Neisseria gonorrhoeae was assessed in Escherichia coli using the Vibrio cholerae toxin B subunit (CtxB) as a heterologous passenger. N-terminal fusions with Iga beta of native CtxB or mutant CtxB protein containing no cysteines were constructed and analysed in isogenic E. coli mutants carrying defects in either or both the ompT (outer membrane protease T) and dsbA (periplasmic disulfide oxidoreductase) determinants. While export of the cystein-less CtxB passenger was independent of the dsbA genotype, the native CtxB passenger was properly translocated across the outer membrane only in the dsbA mutant background. This effect was consistent in the presence and in the absence of the OmpT protease which rather determined the release of surface-bound CtxB into the medium. Therefore, in agreement with previous observations Iga beta-dependent protein secretion requires an unfolded conformation of the passenger domain and can be blocked by disulfide loop formation in the presence of DsbA. Since DsbA acts in the periplasm, this provides evidence for a periplasmic intermediate in the Iga beta-mediated export pathway. E. coli (dsbA ompT) is highly suitable as a strain for the surface display of recombinant proteins via Iga beta, whether or not they contain cysteine residues.

Cellular surface display of dimeric Adx and whole cell P450-mediated steroid synthesis on E. coli.

Bovine adrenodoxin (Adx) was expressed on the surface of Escherichia coli as a monomeric fusion protein with the translocation unit of the AIDA-I autotransporter. The fusion protein remained anchored in the outer membrane by the beta-barrel of the autotransporter. Dimeric Adx molecules were formed spontaneously on the bacterial surface with high efficiencies. Adx dimers could be activated to biological function by chemical incorporation of the [2Fe-2S] cluster. By adding purified adrenodoxin reductase and P450 CYP11A1, a whole cell biocatalyst system was obtained, which effectively synthesized pregnenolone from cholesterol. Addition of artificial membrane constituents or detergents, which was indispensable before to get functional steroidal P450 enzymes, was not necessary. The whole cell activity (0.21 nmol x h(-1) x nmol(-1) CYP11A1) was in the same range as obtained earlier for reconstitution assays. The whole cell system developed here is an easy to handle, stable tool for the expression of membrane-associated P450 enzymes without the need of microsome preparation or reconstitution of artificial membrane vesicles. Moreover, it is the first report on functional dimer formation of a protein anchored on the surface of E. coli after being transported as a monomer. This seems to be a special feature of the autotransporter translocation unit, containing a beta-barrel, motile in the outer membrane and opens a new dimension for the surface display of multimeric proteins.

Development of a simple and rapid assay for the evaluation of inhibitors of human 17α-hydroxylase-C17,20-lyase (P450cl7) by coexpression of P450cl7 with NADPH-cytochrome-P450-reductase in Escherichia coli

P450c17 is a microsomal enzyme catalyzing the last step in androgen biosynthesis. As inhibitors of P450c17 are promising drug candidates for the treatment of prostate cancer, it was our goal to develop a new cellular assay for the in vitro evaluation of potential inhibitors. Human P450c17 was expressed in E. coli and hydroxylase activity was determined using 1,2[ 3 H]-progesterone. As the activity was low (1.7 pmol:min:mg protein), due to a lack of the requisite electron transfer partner NADPH-cytochromeP450-reductase (NADPH-P450-reductase), coexpression of both the enzymes had to be performed. For that purpose, a plasmid was constructed which encoded human P450c17 and rat NADPH-P450-reductase in a transcriptional unit. This strategy led to a 100-fold increase in P450cl7 activity (175 pmol:min:mg protein). Time, pH and temperature dependence of progesterone conversion of this new monooxygenase system was determined. The KM of progesterone was 2.75 mM. An assay procedure for the evaluation of inhibitors was established and modified for high throughput screening using 96-well plates. Selected compounds were tested for their inhibitory activity using this whole cell assay. The data was compared to the results obtained in microsomal testicular preparations.

The Neisseria gonorrhoeae gene aniA encodes an inducible nitrite reductase

The aniA gene of Neisseria gonorrhoeae encodes an outer membrane lipoprotein which is strongly induced when gonococci are grown anaerobically in vitro in the presence of nitrite. Database searches with the amino acid sequence derived from the aniA structural gene revealed significant homologies to copper-containing nitrite reductases from several denitrifying bacteria. We constructed an insertional mutation in the aniA locus of strain MS11 by allelic replacement, to determine whether this locus was necessary for growth in oxygen-depleted environments, and to demonstrate that AniA was indeed a nitrite reductase. The mutant was severely impaired in its ability to grow microaerophilically in the presence of nitrite, and we observed a loss in viability over several hours of incubation. No measurable nitrite reductase activity was detected in the aniA mutant strain, and activity in the strain with a wild-type locus was inducible. Finally, we report investigations to determine whether AniA protein is involved in gonococcal pathogenesis.

Functional Display of Active Bovine Adrenodoxin on the Surface of E. coli by Chemical Incorporation of the [2Fe–2S] Cluster

The display of heterologous proteins on the surface of living cells bears promising options for a wide variety of biotechnological applications. Up to now, however, cellular surface display was merely restricted to simple polypeptide chains. Here we present for the first time the efficient display of a protein (bovine adrenodoxin) that contains an inorganic, prosthetic group in its active form on the surface of Escherichia coli. For this purpose apo‐adrenodoxin was transported to the cell surface and anchored within the outer membrane by the autotransporter pathway. Incorporation of the iron–sulfur cluster was achieved by a single‐vial, one‐step titration under anaerobic conditions. The biological function of surface‐displayed holo‐adrenodoxin could be established through adrenodoxin‐dependent steroid conversion by two different cytochrome P450 enzymes and the number of functional molecules on the cell surface could be determined to be more than 105 per cell. Neither the expression of adrenodoxin nor the incorporation of the chemical iron–sulfur cluster reduced the viability of the bacterial cells.

Autodisplay of Active Sorbitol Dehydrogenase (SDH) Yields a Whole Cell Biocatalyst for the Synthesis of Rare Sugars

Whole cell biocatalysts are attractive technological tools for the regio‐ and enantioselective synthesis of products, especially from substrates with several identical reactive groups. In the present study, a whole cell biocatalyst for the synthesis of rare sugars from polyalcohols was constructed. For this purpose, sorbitol dehydrogenase (SDH) from Rhodobacter sphaeroides, a member of the short‐chain dehydrogenase/reductase (SDR) family, was expressed on the surface of Escherichia coli using Autodisplay. Autodisplay is an efficient surface display system for Gram‐negative bacteria and is based on the autotransporter secretion pathway. Transport of SDH to the outer membrane was monitored by SDS‐PAGE and Western blotting of different cell fractions. The surface exposure of the enzyme could be verified by immunofluorescence microscopy and fluorescence activated cell sorting (FACS). The activity of whole cells displaying SDH at the surface was determined in an optical test. Specific activities were found to be 12 mU per 3.3×108 cells for the conversion of D‐glucitol (sorbitol) to D‐fructose, 7 mU for the conversion D‐galactitol to D‐tagatose, and 17 mU for the conversion of L‐arabitol to L‐ribulose. The whole cell biocatalyst obtained by surface display of SDH could also produce D‐glucitol from D‐fructose (29 mU per 3.3×108 cells).

Functional esterase surface display by the autotransporter pathway in Escherichia coli

Bacterial surface display is a promising tool for a wide variety of biotechnological applications. In this work, a carboxylesterase, EstA from Burkholderia gladioli was translocated to the surface of Escherichia coli using the autotransporter pathway. For this purpose, an artificial gene was constructed by PCR, that encodes a fusion protein of EstA and the essential autotransporter domains. Esterase activity of whole cells expressing the EstA-autotransporter fusion protein could be detected by a filter overlay assay using α-naphthylacetate as substrate as well as by an agar plate pH-assay using p-nitrophenylacetate as a substrate. The specific esterase activity of whole cells was determined to be 1.7 mU/mg protein with p-nitrophenylacetate. After differential cell fractionation, the specific esterase activity of the outer membrane fraction was determined to be 23 mU/mg protein, using the same substrate. Western blot analysis of the different cell fractions with an EstA specific antibody yielded positive signals only in the outer membrane fraction. Furthermore, the detected protein band was in the correct size, as it was predictable from the amino acid sequence of the fusion protein. In activity staining of SDS-gels using α-naphthylacetate as a substrate, it was the identical band that exhibited esterase activity. Surface exposure could be demonstrated by proteinase K digestion of the esterase domain, whereby the protease was externally added to intact cells. These results indicated that EstA is targeted to the surface of E. coli by the autotransporter pathway in its active form.

A carbon nanotube metal semiconductor field effect transistor-based biosensor for detection of amyloid-beta in human serum.

We have developed a carbon nanotube (CNT) film-based biosensor with a metal semiconductor field effect transistor structure (MESFET). A gold top gate was deposited on the middle of the CNT channel and probe antibodies were immobilized on the gold top gate with an antibody-binding protein, protein G or Escherichia coli outer membrane (OM) with autodisplayed Z-domains of protein A. These CNT-MESFET biosensors exhibited a higher sensitivity than the CNT-FET biosensor with probe antibodies immobilized using a chemical linker, since the orientation of immobilized antibodies was controlled by the antibody-binding proteins. In addition, nonspecific binding was effectively inhibited by E. coli OM. Using the CNT-MESFET biosensors with E. coli OM containing Z domain, we detected amyloid-β (Aβ) in human serum, one of the biomarkers for early diagnosis of Alzheimers disease. Aβ at the level of 1 pg/mL in human serum could be measured in real-time and without labeling, which was lower than a limit of detection for plasma Aβ using an enzyme-linked immune sorbent assay. These results suggested that our CNT-MESFET biosensors might be applicable for an early diagnosis of Alzheimers disease.