Immobilized Receptor Research Articles

Field Deployable Sensor Arrays with Molecular Selectivity for Soil Health

Monitoring soil health is very important in agriculture. Soil health can be monitored by the sensing the metabolomic activities of microbiota as increased concentrations in CO2, CH4, N2O, vapors in the soil. A vapor sensor based on microfabricated cantilever array is an ideal sensor platform for field deployable miniature sensors. Many such platforms, such as cantilever arrays, have been demonstrated as sensitive sensors for chemical and biological detection. In fact, the array-based sensor platform satisfies almost all of the requirements of an ideal high-performance sensor, such as its very high sensitivity, miniature size, array based multi-analyte detection, and low power consumption. However, poor molecular selectivity, especially for small molecules at trace level, as well as the lack of reproducibility, pose formidable challenges in translating this highly sensitive sensor platform into a practical reality. Traditionally, selectivity in chemical and biological sensing is achieved by using immobilized receptors or chemical interfaces on sensor surfaces. While poor selectivity in small molecule detection using reversible receptors is due to the lack of uniqueness of receptor-analyte interaction, nonuniformity in the immobilized receptor graft density is the reason for unacceptable rates of reproducibility in these sensors. We have used a multi-modal, multi-physics approach in overcoming these challenges. Multi-modal data when analyzed using deep learning techniques, show enhanced selectivity, sensitivity, and reliability. In order to operate these sensors in a field-scale study, a long range through-the-soil (TTS) wireless power transfer method is developed. Energy from an adjacent solar panel array is distributed wirelessly via conduction currents that propagate outward along and through the soil. Power transfer within a 0.25-ha (0.6acre) area is demonstrated using an average input power of 6W.This research is supported by NSF-SitS Award Nos. 2226614 and 2226612

Research on drug-receptor interactions and prediction of drug activity via oriented immobilized receptor capillary electrophoresis.

Oriented covalent immobilized β2 -adrenergic receptor (β2 -AR) CE (OIRCE) was developed to determine the interactions between a set of natural extracts of Radix Paeoniae Rubra (NERPR) and β2 -AR, and to predict the activity of NERPR. The inner capillary surface is chemically bonded with stable β2 -AR coating via microwave-assisted technical synthesis. The modified capillaries were characterized via infrared spectroscopy and fluorescence microscopy. Furthermore, the bonding amounts of β2 -AR were first obtained via fluorescence spectroscopy method. In determining the amount of bonded β2 -AR, the regression equation A = 576707C+35.449 and the correlation coefficient 0.9995 were obtained. This result revealed an excellent linear relationship in the range of 2×10(-4) mg/mL to 1×10(-3) mg/mL. The normalized capacity factor (KRCE ) was obtained using OIRCE in evaluating drug-receptor interactions. Related theories and equations were used to calculate KRCE values from apparent migration times of a solute and EOF. The order of KRCE and the binding constant (Kb ) values between drugs and β2 -AR was well consistent. The results confirmed that the OIRCE and KRCE values can be effectually used to investigate drug-receptor interactions, and OIRCE has the potential to predict drug activity and to select leading compounds from natural chemicals.

Immobilised histidine tagged β2-adrenoceptor oriented by a diazonium salt reaction and its application in exploring drug-protein interaction using ephedrine and pseudoephedrine as probes.

A new oriented method using a diazonium salt reaction was developed for linking β 2-adrenoceptor (β 2-AR) on the surface of macroporous silica gel. Stationary phase containing the immobilised receptor was used to investigate the interaction between β 2-AR and ephedrine plus pseudoephedrine by zonal elution. The isotherms of the two drugs best fit the Langmuir model. Only one type of binding site was found for ephedrine and pseudoephedrine targeting β 2-AR. At 37 °C, the association constants during the binding were (5.94±0.05)×103/M for ephedrine and (3.80±0.02) ×103/M for pseudoephedrine, with the binding sites of (8.92±0.06) ×10−4 M. Thermodynamic studies showed that the binding of the two compounds to β 2-AR was a spontaneous reaction with exothermal processes. The ΔGθ, ΔHθ and ΔSθ for the interaction between ephedrine and β 2-AR were −(22.33±0.04) kJ/mol, −(6.51±0.69) kJ/mol and 50.94±0.31 J/mol·K, respectively. For the binding of pseudoephedrine to the receptor, these values were −(21.17±0.02) kJ/mol, −(7.48±0.56) kJ/mol and 44.13±0.01 J/mol·K. Electrostatic interaction proved to be the driving force during the binding of the two drugs to β 2-AR. The proposed immobilised method will have great potential for attaching protein to solid substrates and realizing the interactions between proteins and drugs.

Abstract 279: Use of GFP-STAT3βtc for EMSA analysis of protein-protein and protein-DNA interactions in tumorigenic signalling pathways

Abstract STAT3 (Signal transducer and activator of transcription 3) is a homodimeric transcription factor that binds to the response element of a number of genes that promote tumorigenesis when over-expressed. Tumorigenesis, in many cases, correlates with an aberrant signalling cascade that leads to uncontrolled activation of STAT3. Activation is widely understood to be a result of phosphorylation at tyrosine 705 which leads to dimerisation and DNA binding. We have investigated the dimerisation and DNA binding of STAT3βtc in an isolated cell-free system in order to understand the conditions which modulate these interactions, and to facilitate the discovery of small molecules capable of interfering with these interactions. We designed a GFP-STAT3βtc construct in which the N-terminal 126 amino acids of STAT3 were replaced with spectral variants of the GFP (green fluorescent protein) to act as a reporter. The constructs also lacked the unstructured C-terminal activation domain of STAT3 to avoid aggregation of the purified protein. Phosphorylated and non-phosphorylated GFP-STAT3βtc proteins were purified from BL21(DE3)TKB1 and Rosetta strains of E. Coli, respectively. Soluble expression was observed when tagged with GFP at the N-terminus but not the C-terminus. Using both conventional ELISA and fluorescence-based microtiter plate assays, we demonstrated that both active (phosphorylated) and inactive (non-phosphorylated) fluorescent STAT3βtc can bind to immobilised dsDNA (M67; a modified c-fos sis inducible enhancer) or an immobilised surrogate receptor (a phosphotyrosyl peptide derived from the interleukin-6 receptor subunit gp130), respectively. Furthermore, we were able to demonstrate disruption of a pre-formed non-phosphorylated GFP-STAT3βtc/DNA complex with a phosphotyrosyl peptide at an IC50 of 30 µM. In contrast, 500 µM peptide appeared to have no effect on phosphorylated GFP-STAT3βtc bound to M67. In summary, we have developed an EMSA assay for the STAT3/STAT3 transcription factor based on a protein mobility shift rather than a DNA mobility shift, and which utilizes fluorescent tagged STATs. Both non-phosphorylated and phosphorylated fluorescent STAT3 proteins form a stable complex with M67 DNA, but only the non-phosphorylated STAT3/DNA complex can be disrupted with a phosphotyrosyl peptide in the EMSA assay. This assay is now being utilized for the discovery of novel small molecule inhibitors of the STAT3/STAT3 interaction. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 279. doi:10.1158/1538-7445.AM2011-279

Silica-attached molecular receptor complexes for benzoates and naphthoates

AbstractA series of cyclen (1,4,7,10-tetraazacyclododecane) derived molecular receptors for aromatic oxoanions, that are activated by complexation with Cd(II), have been covalently linked to 3-(glycidoxy)propyl-functionalised silica gel (70-230 mesh). These immobilised receptor complexes are highly effective for the sequestration of o-hydroxybenzoates and 2-naphthoate from aqueous solution, achieving a >80% saturation level by stirring the material in the aqueous solution for 1 h at pH 7.00 and 298 K. Examination of the uptake levels of a variety of different benzoates and naphthoates suggests that the retention mechanism involves a combination of classical hydrogen bonding and non-classical, water mediated, O-H···π hydrogen bonding. Contrary to expectations, attachment of hydroxy terminated polyether chains to the periphery of the receptor complex diminished the level of uptake.Graphical synopsisSilica-attached receptor complexes of the type shown are effective materials for sequestering o-hydroxybenzoates from aqueous solution. [IMAGE]

Label-free DNA detection platform based on atomic force microscopy visualisation: characterising the molecular-recognition-triggered conformational changes of an immobilised receptor oligonucleotide probe

Using atomic microscopy imaging, probe DNA sequence self-assemblies developed on Si(100) substrates undergo a conformational transition from an extended stem-loop structure to a double helix; such assemblies readily report on DNA molecular recognition events and should be suitable as a label-free, DNA hybridisation assay platform.

The application of polythiol molecules for protein immobilisation on sensor surfaces

The immobilisation of bio-receptors on transducer surfaces is a key step in the development of biosensors. The immobilisation needs to be fast, cheap and most importantly should not affect the biorecognition activity of the immobilised receptor. The development of a protocol for biomolecule immobilisation onto a surface plasmon resonance (SPR) sensor surface using inexpensive polythiol compounds is presented here. The method used here is based on the reaction between primary amines and thioacetal groups, formed upon reaction of o-phthaldialdehyde (OPA) and thiol compounds. The self-assembled thiol monolayers were characterised using contact angle and XPS. The possibility to immobilise proteins on monolayers was assessed by employing BSA as a model protein. For the polythiol layers exhibiting the best performance, a general protocol was optimised suitable for the immobilisation of enzymes and antibodies such as anti-prostate specific antigen (anti-PSA) and anti Salmonella typhimurium. The kinetic data was obtained for PSA binding to anti-PSA and for S. typhimurium cells with a detection limit of 5 × 10 6 cells mL −1 with minimal non-specific binding of other biomolecules. These findings make this technique a very promising alternative for amine coupling compared to peptide bond formation. Additionally, it offers opportunity for immobilising proteins (even those with low isoelectric point) on neutral polythiol layers without any activation step.

Microfluidic competition assay via equilibrium binding

Competitive binding of analytes in solution, to an immobilized receptor, is a preferred method for quantifying molecules in complex mixtures. We have developed a theoretical and experimental framework for the microfluidic competition assay. A mathematical model describes the transient, convection–dispersion of solutes, undergoing equilibrium binding to immobilized receptors, while entrained in a low Reynolds number incompressible fluid flowing through a microchannel. The proposed method involves monitoring of the elution profile of a reference molecule and ligand in the presence of a competitor. The time difference between the two breakthrough curves provides a measure of the unknown concentration of the competitor. Theoretical results illustrate the general method for determining the equilibrium dissociation constant ( K d) of the ligand and competitor, as well as the competitor concentration. Experimental data is presented for the binding of fluorescein labeled insulin and unlabeled insulin to a monoclonal antibody. It is found that the unlabeled insulin binds with higher affinity ( K d = 0.17 μM) than the labeled insulin ( K d = 0.76 μM). The potential advantages of the method and further improvements in the model are discussed.

New reactive polymer for protein immobilisation on sensor surfaces

Immobilisation of biorecognition elements on transducer surfaces is a key step in the development of biosensors. The immobilisation needs to be fast, cheap and most importantly should not affect the biorecognition activity of the immobilised receptor. A novel protocol for the covalent immobilisation of biomolecules containing primary amines using an inexpensive and simple polymer is presented. This tri-dimensional (3D) network leads to a random immobilisation of antibodies on the polymer and ensures the availability of a high percentage of antibody binding sites. The reactivity of the polymer is based on the reaction between primary amines and thioacetal groups included in the polymer network. These functional groups (thioacetal) do not need any further activation in order to react with proteins, making it attractive for sensor fabrication. The novel polymer also contains thiol derivative groups (disulphide groups or thioethers) that promote self-assembling on a metal transducer surface. For demonstration purposes the polymer was immobilised on Au Biacore chips. The resulting polymer layer was characterised using contact angle meter, atomic force microscopy (AFM) and ellipsometry. A general protocol suitable for the immobilisation of bovine serum albumin (BSA), enzymes and antibodies such as polyclonal anti-microcystin-LR antibody and monoclonal anti-prostate specific antigen (anti-PSA) antibody was then optimised. The affinity characteristics of developed immunosensors were investigated in reaction with microcystin-LR, and PSA. The calculated detection limit for analytes depended on the properties of antibodies. The detection limit for microcystin-LR was 10 ng mL −1 and for PSA 0.01 ng mL −1. The non-specific binding of analytes to synthesised polymers was very low. The polymer-coated chips were stored for up to 2 months without any noticeable deterioration in their ability to react with proteins. These findings make this new polymer very promising for the development of low-cost, easy to prepare and sensitive biosensors.

Scanning force microscopy based amperometric biosensors

Cantilever-based biosensing has developed into an important research area especially for biomedical and clinical analysis. In particular, the possibility to scan miniaturized amperometric biosensors with high-fidelity distance control across biomedically relevant sample surfaces enables the determination of pertinent analytes such as, e.g., adenosine 5′-triphosphate (ATP). ATP is involved in a wide variety of important regulatory cellular mechanisms, and quantification of ATP has therefore been the focus of extensive research in recent years. Usually, scanning force microscopy-based biosensing relates to detection principles based on either mass-sensitive mechanical transduction, which can be detected as a frequency change of the cantilever, or a change in the force constant due to the absorption of molecules onto the sensor surface, as shown in Fig. 1a. Alternatively, a bimetallic cantilever can be used as a temperature sensing device for detecting calorimetric changes due to specific binding to an immobilized receptor, or due to absorption of molecules. Advanced microfabrication in combination with sophisticated surface functionalization schemes results in sensing interfaces tailored to specifically interact with analyte molecules, thus providing specificity and sensitivity of such miniaturized devices to the picomolar and even femtomolar concentration range [1]. Conventional cantilevertype biosensors do not provide a sharp tip, and are typically not applied for laterally resolved measurements in an imaging mode, which requires that the sensor is scanned across, e.g., a biological specimen at a controlled distance. Recently, the National Heart Lung and Blood Institute has published a report identifying demands for future developments related to diagnosis and therapy of cardiovascular, pulmonary, and hematologic diseases, which identifies in vivo nanosensors as a promising prospective application of nanotechnology for real-timemonitoring of biological signals in response to cardiac or inflammatory events [2]. Hence, besides measurements in complex background matrices, the analytical challenges encompass the ability of biosensors to provide information on structural changes along with chemical information on, e.g., regulatory processes at single cells or cell ensembles, ideally at a nanometer scale. Consequently, miniaturization of the transducer and sensing interface, along with positioning of such miniaturized sensors in close proximity to the sample surface providing laterally resolved quantitative information is a prerequisite. Alternatively to mass-sensitive cantilever-based sensing in bulk solution, atomic force microscopy (AFM) probes can be chemically functionalized, which enables detection and quantification of forces associated with single-molecule binding events at ambient conditions. Recently, this concept has evolved into a combined tool providing laterally resolved information along with force mapping, also known as topography and recognition imaging [3], which enables the visualization of single receptor binding sites at biological surfaces. The present article, however, focuses on the detection of chemical signaling events involving the secretion of transmitter molecules into the extracellular space, and associated efforts to detect such molecules and their concentration for enhancing fundamental understanding on disease-related cellular processes at a molecular level. Anal Bioanal Chem (2008) 390:239–243 DOI 10.1007/s00216-007-1670-8

Engineering and functional immobilization of opioid receptors

Opioid receptors, like many G protein-coupled receptors (GPCRs), are notoriously unstable in detergents. We have now developed a more stable variant of the mu-opioid receptor (MOR) and also a method for the immobilization of solubilized, functional opioid receptors on a solid phase (magnetic beads). Starting with the intrinsically more stable kappa-opioid receptor (KOR), we optimized the conditions (i.e. detergents and stabilizing ligands) for receptor extraction from lipid bilayers of HEK293T cells to obtain maximal amounts of functional, immobilized receptor. After immobilization, the ligand binding profile remains the same as observed for the membrane-embedded receptor. For the immobilized wild-type mu-opioid receptor, however, no conditions were found under which ligand binding capacity was retained. To solve this problem, we engineered the receptor chimera KKM where the N-terminus and the first transmembrane helix (TM1) of wild-type MOR is exchanged for the homologous receptor parts of the wild-type KOR. This hybrid receptor behaves exactly as the wild-type MOR in functional assays. Interestingly, the modified MOR is expressed at six times higher levels than wild-type MOR and is similarly stable as wild-type KOR after immobilization. Hence the immobilized MOR, represented by the chimera KKM, is now also amenable for biophysical characterization. These results are encouraging for future stability engineering of GPCRs.

Application of an SPR-based receptor assay for the determination of biologically active recombinant bone morphogenetic protein-2

Human bone morphogenetic protein-2 (hBMP-2) is a member of the human transforming growth factor-beta superfamily. Biologically active bone morphogenetic protein-2 (BMP-2) is a dimeric protein that binds in the first step of the signal transduction cascade to specific receptors on the cell-surface. This specific interaction of the dimeric protein with the extracellular ligand-binding domain (ECD) of the receptor was used to develop a receptor-based assay based on an optical biosensor system (Biacore 2000, Biacore AB, Uppsala, Sweden). The ECD of the BMP-receptor type IA, tagged with the Fc part of IgG (BMPR-IA-Fc), was immobilised on the surface of a dextran-protein A-coated sensor chip. Calibration curves were obtained with purified and biologically active recombinant hBMP-2 (rhBMP-2) that showed a linear range from approximately 5 to 250 nM rhBMP-2. Moreover, this assay was used to quantitatively follow the generation of biologically active protein during the renaturation from unfolded and reduced monomers to biologically active dimers. A refolding mixture containing renatured dimeric rhBMP-2 and not correctly folded monomers, was used as the sample solution without any further pre-treatment. It was proven that only the biologically active dimers were recognised by the immobilised receptor, so the generation of biologically active rhBMP-2 during the renaturation process could be monitored directly and rapidly. Furthermore, the results from the optical sensor obtained during the renaturation process showed a good correlation with the data obtained by non-reducing SDS-PAGE analysis carried out at the end of the renaturation process. These data show that the disulphide-bonded dimer corresponds to the biologically active protein capable of binding the BMP-receptor type IA.

My life with affinity

I was born in 1935 in Warsaw, Poland, to a rabbinical family. My early childhood occurred during World War II in German-occupied Poland and then in Russia. My father was killed in the concentration camp of Flossenberg; my mother, my sister and I survived and emigrated from Germany to Israel in 1949. In Israel I finished my primary school and high school. I completed my B.Sc. in chemistry and physics at Bar-Ilan University and my Ph.D. in biochemistry at the Weizmann Institute of Science.

CD8 alpha-deficient mice are highly susceptible to 5-fluorouracil-induced lethality.

Intestinal intraepithelial lymphocytes (i-IEL) expressing CD8 alpha are located in the intestine and may confer protection against invasion of intestinal microflora. We found that mice rendered deficient in CD8 alpha molecules by homologous recombination were susceptible to 5-fluorouracil (5-FU)-induced lethality accompanied by translocation of members of the enterobacteria. The number of i-IEL was greatly reduced on day 6 after 5-FU administration in both CD8 alpha(+/-) mice and CD8 alpha(-/-) mice, whereas the recovery of the level of i-IEL thereafter was significantly impaired in CD8 alpha(-/-) mice compared with that in CD8 alpha(+/-) mice. The ability of i-IEL to produce gamma interferon in response to immobilized T-cell receptor (TCR) alpha beta or TCR gamma delta monoclonal antibodies was significantly lower in CD8 alpha(-/-) mice than in CD8 alpha(+/-) mice. Transfer of CD8(+) i-IEL conferred significant protection against 5-FU-induced lethality in CD8 alpha(-/-) mice. The results suggest that CD8(+) i-IEL play an important role in protection against 5-FU-induced lethality with translocation of Enterobacteriaceae.

Receptor binding protein amperometric affinity sensor for rapid β-lactam quantification in milk

Screen-printed devices, incorporating working electrode immobilised β-lactam specific receptor binding protein, were employed to measure penicillin G levels in milk. Quantification was achieved through ELISA-based affinity-assay format coupled to amperometric determination of bound enzyme label activity. Assay inhibition increased from zero, in the absence of penicillin G in milk, to 33.5 and 77.1% reduction in signal response in the presence of 5 μg kg −1 and 10 μg kg −1 penicillin G, respectively. The maximum residue limit of penicillin G in milk for consumption is 5 μg kg −1, as defined by the FDA. Coefficient of variation values varied from 4.2–26.4%. The assay incorporates a 2–4 min incubation step, a rapid washing step and 1–2 min measurement step. The receptor binding protein is specific for the major β-lactam antibiotic types. The assay is simple to perform and requires minimum reagent usage, making it ideal as a field-based screening tool for β-lactam quantification in milk.

Measurement of Total and Bioactive Interleukin-2 in Tissue Samples by Immunoaffinity-Receptor Affinity Chromatography

The detection and measurement of cytokines is an important issue in the clinico-pathological diagnosis of several clinical entities, including organ transplant rejection. Existing techniques, although sensitive, measure only total cytokine concentrations and cannot measure bioactivity. A chromatographic system combining immunoaffinity chromatography with an immobilized receptor detection cartridge has been developed for measuring total and bioactive interleukin (IL)-2 concentrations in tissue extracts prepared from biopsy materials taken from renal transplant recipients during both rejection and drug-induced nephrotoxic episodes. The technique employs a short high-pressure chromatography column packed with antibody-coated glass beads for the initial analyte separation and concentration, followed by detection of bioactive molecules through their interactions with specific, immobilized receptors. This system compares favourably with both conventional bioassays and immunoassays for measuring IL-2 in tissue samples.

Development of a Novel TNFα Ligand-Receptor Binding Assay for Screening NatchemTM Libraries

The TNFα receptor is a major therapeutic target since over production of TNFα plays a key role in the development of septic shock following bacterial infection and has been implicated in the pathogenesis of many inflammatory disorders such as rheumatoid arthritis. To screen our NATCHEMTM Libraries for novel natural product inhibitors of this target we have developed a sensitive immobilised TNFα receptor binding assay based on a commercially available recombinant soluble TNFα receptor (p55) and [125I]-TNFα.

Mathematical Analysis of Sensors Based on Affinity Interactions between Competitive Receptor-Protein Pairs

Affinity sensors with detection principle of competitive displacement of a responsive protein from an immobilised receptor by an analyte were subject to mathematical analysis. The model considered two competing binding reactions between the analyte and the responsive protein for the immobilised receptor in the solid phase. A cubic algebraic equation generated from the analysis was solved to get theoretical calibration curves for the sensor relating the signal generated, which is the bulk concentration of the responsive protein detected in solution, to the analyte concentration. The simulated results indicated several important system variables to consider when optimising the sensor's performance. They are the concentration of the responsive protein initially loaded, the density of the immobilised receptor, and the relative binding strength of both the analyte and the responsive protein toward the receptor. To test this model, experiments were carried out with a hypothetical sensor system, using bovine chymosin as the responsive protein, pepstatin-agarose as the immobilised receptor, and pepsin as the analyte. The model in general can describe the behavior of this type of affinity sensor reasonably well. From kinetic study of chymosin displacement in the model system, the effect of binding strength between responsive protein and receptor on the response of the sensor was also discussed.

Femtomolar sensitivity using a channel-etched thin film waveguide fluoroimmunosensor

A dual channel, evanescent fluoroimmunoassay format is used to detect femtomolar analyte concentrations (i.e. less than 1 part per trillion [w/w]) on an etched channel siliconoxynitride thin film integrated optical waveguide. Two assays are used to demonstrate the dose-response behaviour of the sensor: (1) a direct assay of a fluorescently-labeled protein ligand binding to an immobilized protein receptor, and (2) an indirect sandwich assay of a non-fluorescent protein ligand binding to an immobilized protein receptor, as detected by the binding of a fluorescently-labeled secondary receptor protein. A red-emitting cyanine dye (Cy-5), which minimized background fluorescence and scatter losses of the waveguide, was used in both assays. To our knowledge, this is the first report of femtomolar sensitivity in an immunosensing instrument.

The Escherichia coli G-fimbrial lectin protein participates both in fimbrial biogenesis and in recognition of the receptor N-acetyl-D-glucosamine.

The gafD gene encoding the N-acetyl-D-glucosamine-specific fimbrial lectin (adhesin) protein GafD of uropathogenic Escherichia coli was cloned and subjected to genetic analysis. The corresponding gene product was isolated as a MalE fusion protein. The lectin gene was identified with the aid of deletion mutagenesis; mutations in gafD impaired either receptor binding or both receptor binding and fimbria production, depending on the mutation created. All mutants converted to wild-type expressors when complemented in trans with the cloned intact gafD gene. The predicted 354-amino-acid sequence of GafD, deduced from the nucleotide sequence, is closely related to those of the fimbria-associated F17-G and F17b-G proteins coded for by enterotoxigenic and invasive E. coli strains. Isolated GafD was shown to recognize N-acetyl-D-glucosamine by virtue of specific binding to an immobilized receptor, thus proving directly that GafD is a sugar-binding protein. Our results indicate that GafD as such is sufficient for receptor recognition and that the protein also participates in fimbrial biogenesis.