S-layer proteins are considered as a good nanocarrier due to their binding and self-assembled properties. These can be used to prepare the immunomatrixes for the removal of toxins from the samples.
S-layer proteins are considered as a good nanocarrier due to their binding and self-assembled properties. These can be used to prepare the immunomatrixes for the removal of toxins from the samples.
Two S-layer proteins 70 and 40 kDa of thermophilic Thermobifida fusca were extracted with guanidine hydrochloride and purified. Antibodies against S-layer proteins were developed, and their monospecificity was checked. Immunogold labelling indicated that these are surface proteins. Immunomatrixes (70-SLIM, 40 SLIM) were prepared by covalently immobilizing S-layer proteins in microwell and further conjugated with anti- Staphylococcus aureus enterotoxin B (SEB) antibodies. The binding of 70 and 40 kDa proteins was observed nearly 7·0 μg cm−1 to binding area, and the conjugation with anti-SEB antibodies was found 1·22 μg μg−1 of 70 kDa and 0·875 μg μg−1 of 40 kDa. The average binding and elution of pure SEB toxin on 70-SLIM and 40-SLIM was 5·0 μg SEB toxin. The SEB toxin in milk samples was also removed on immunomatrixes successfully.
It is the first report, and this study shows that the thermophilic S-layer proteins can be used to prepare the immunomatrixes.
Information in this study can be used to design the strategies for the removal of biologically important materials or toxins from samples.
Most common surface structures on archaea and bacteria are monomolecular crystalline arrays of proteinaceous surface layers or S-layers that have oblique, square or hexagonal lattice symmetry (Sleytr 1978; Sleytr et al. 1993, 1999). The lattice structure of S-layers consists of one, two, three, four or six identical (glyco) protein subunits that have space 2·5–35 nm in range. S-layers are 5–25 nm thick with smooth outer surface and more corrugated inner surface. S-layers also have two or more pores (2–8 nm in range) which are identical in size and morphology and occupy up to 70% of surface area (Messner et al. 1984; Phipps et al. 1991; Peters et al. 1995, 1996).
S-layer proteins have been tested in nanobiotechnological research due to self-assembled property. The smooth outer surface and corrugate inner face is important for orientation of S-layer on solid surface. The bottom-up strategies for S-layer proteins is useful to provide uniform nanostructure as well as generating multilayered supramolecular assemblies. S-layer proteins have also been used for the preparation of affinity microparticles (Weiner et al. 1994; Herak, et al. 1990), for the development of dipstick solid immunoassays (Breitwieser et al. 1998), immobilization of enzymes (Küpcü et al. 1995; Sleytr et al. 1999), development of biosensors (Neubauer et al. 1993, 1994, 1997; Kauffmann 2000) and for vaccine development (Beveridge and Koval, 1993; Malcolm et al. 1993; Messner 1997; Yoneda et al. 2003).
The amino acid analysis of S-layer proteins revealed that S-layer proteins of organisms from different phylogenetic branches have nearly same composition with minor difference (Sleytr and Messner 1983; Beveridge 1994; Sara and Sleytr 1996; Sleytr et al. 1996) and possess high content of acidic and hydrophobic amino acids. Lysine is a dominant basic amino acid while arginine, histidine and methionine contents are low. Cysteine was detected in a few S-layer proteins (Messner and Sleytr 1992; Sara and Sleytr 1996). Different methods are available to detach the S-layers and its disintegration into protomeric units (Koval 1988; Sara and Sleytr 1989; Messner and Sleytr 1992; Beveridge 1994; Sleytr et al. 1996). S-layer proteins of gram-negative bacteria can be isolated by using metal-chelating agents or cation substitution agents. The isolated S-layer subunits have ability to recrystalize into regular arrays in suspension or on solid surfaces (Sleytr 1975; Beveridge 1994; Sara and Sleytr 1996; Pum and Sleytr 1999).
S-layers represent the outermost interaction zone with the respective environment and their functions vary from species to species. In archaea, S-layer is the cell wall component and is important for mechanical stabilization. Additional functions associated with S-layers are protection from bacteriophages and phagocytosis, resistance against low pH, barrier against high molecular weight substances, protection from lytic enzymes, protection and stabilization of internal organelles from hazardous environments (Sleytr et al. 1997). Some pathogenic bacteria such as Corynebacterium diphtheria, Bacillus anthracis and some microflora of mouth have S-layers which make them hydrophobic and protect from phagocytosis. (Sleytr et al. 1997). S-layer proteins of some bacteria such as Th. Thermohydrosulfuricus LIII-69 and Bacillus sepharecus CCM 2120 was used as nanocarrier for immobilization and diagnostic purposes (Weiner et al. 1994; Grogono et al. 2002).
In the present study, S-layer proteins of thermophilic Thermobifida fusca were purified and characterized. The purified protein was used for the development of immunomatrix assay for the removal and detection of Staphylococcus aureus SEB antigen in the samples.
Thermobifida fusca was obtained from School of Biological Sciences, University of the Punjab, Lahore and maintained on Hager Dahl medium under aerobic conditions. The final concentration of ingredients in the medium was as follow, 3·1 g l−1 (NH4)2SO4; 1·0 g l−1 cellulose; 2·0 g l−1 glucose; 1·5 g l−1 NaCl; 0·9 g l−1 KH2PO4; 0·91 g l−1 K2HPO4 and micronutrients 0·200 g l−1 MgSO4∙7H2O; 0·008 g l−1 ZnSO4∙7H2O; 0·02 g l−1 FeSO4∙7H2O; 0·015 g l−1 MnSO4∙H2O; 0·026 g l−1 CaCl2 (Sunil et al., 2011). 50 ml of the medium was inoculated with T. fusca and placed in an orbital shaker at 48°C for 72 h.
Ten millilitre of overnight culture of T. fusca was used to inoculate 500 ml of fresh medium (described above) and cultured it until OD600 reached 0·7. Cells were harvested at 30 000 g for 30 min at 4°C. Cells were treated according to the procedure as described with little modification (Boot et al. 1993). Cell pellet was resuspended in 3·0 ml of extraction buffer (4·0 mol l−1 guanidine hydrochloride in 20 mmol l−1 Tris-Cl, pH 7·5) and kept for 1 h at 37°C with mild shaking. The cell suspension was centrifuged at 8000 g for 25 min at 4°C. The supernatant was separated and dialysed against 50 mmol l−1 Tris-Cl buffer pH 7·5. The dialysed supernatant was again centrifuged at 30 000 g for 20 min at 4°C. The residual pellet was discarded, and supernatant was used for ion-exchange chromatography.
Total cell lysate of T. fusca was prepared as follows: 0·5 gm of cells was suspended in 2·0 ml of crushing buffer (extraction buffer + 1·0% Tween 20) and sonicated for five times (10 burst per times, Soni-Prep). The sonicated cells were further homogenized in Daunce homogenizer for 1–2 min. The lysate was centrifuged at 30 000 g for 20 min at 4°C. The supernatant was used for indirect ELISA.
The dialysed supernatant was loaded on DEAE-cellulose ion-exchange column (1·5 × 10 cm) equilibrated with two-column volume of Q-buffer (8·0 mol l−1 urea; 50 mmol l−1 Tris-Cl, pH 7·5). The dialysed supernatant was loaded at a flow rate of 1·0 ml min−1. After washing with two column volume of Q-buffer, the protein fractions were eluted with stepwise gradient (0·1, 0·2, 0·3 and 0·4 mol l−1 NaCl in Q-buffer) at a flow rate of 0·5 ml min−1 by monitoring the peak fraction absorbance at 280 nm in Econo UV monitor (Bio-Rad, CA, USA). Peak fractions were pooled, and the protein concentration was determined by dye-binding method using bovine serum albumin as standard (Bradford 1976).
The pooled protein fractions from ion-exchange column were fractionated onto gel filtration column packed with Sephadex G-75 resin. The column was washed with two columns of Q-buffer, and the flow rate was adjusted 1·0 ml min−1. After equilibration, the fractions from ion exchange were loaded onto the column, and peak fractions were collected at a flow rate of 0·5 ml min−1. The peak fractions were pooled and concentrated in spin column (cut-off, 10 kDa) according to given instructions (Sigma-Aldrich, St Louis, USA). The protein concentration was measured by Bradford reagent using bovine serum albumin as standard (Bradford 1976). The protein concentration was adjusted to 1·0 mg ml−1, and the molecular weight was determined on 10% SDS-PAGE (Laemmli 1970).
Antibodies against purified 70 and 40 kDa S-layer proteins were developed in male Balb/C. Both 70 and 40 kDa S-layer proteins were mixed with Freund's complete adjuvant in 1 : 1 ratio separately to immunize the mice subcutaneously (60–70 μg protein per mouse per time) separately. Total five injections were given at 14 days intervals. Whole blood was isolated by cardiac puncture and serum was separated. The monospecific antibodies were purified on affinity for resin prepared by conjugation of purified 70 and 40 kDa S-layer proteins with CN-Br activated sepharose 4B separately (Harlow and Lane 1988). The purified antibodies were stored at −20°C till further process.
Indirect enzyme-linked immunosorbent assay (iELISA) was used to detect anti-70 kDa and anti-40 kDa antibodies in mouse sera by using purified 70 and 40 kDa and total cell lysate as coating antigens. Rabbit anti-mouse IgG-alkaline phosphatase conjugated was added in wells to bind with antigen–antibody complex. The colour reaction was developed by using substrate (0·01 mol l−1 paranitrophenyl phosphate in 0·05 mol l−1 ethanolamine buffer, pH 8). The monospecificity of anti-70 kDa and anti-40 kDa antibodies for S-layer proteins (70- and 40-kDa) was confirmed by Western blot analysis (Towbin et al. 1979). The reactivity of both antibodies with total cell lysate was also checked.
Colloidal gold particles (5–10 nm) were prepared by the reduction of chloroauric acid. Briefly, 100 mg of gold salt was dissolved in 1000 ml of deionized water and boiled for 30 min. During boiling, 12·5 ml of 1% trisodium citrate was added under vigorous stirring. The colour of the solution turned from yellow to dark blue and finally to orange-red. The solution was cooled down to room temperature, and the pH 7·5 was adjusted with 0·1 mol l−1 K2CO3. The final colour of the solution was deep red.
Affinity-purified anti-70 kDa and anti-40 kDa antibodies were conjugated with gold nanoparticles. The amount of antibodies required to stabilize the gold solution was determined by flocculation test. In flocculation test, two-fold serial dilutions of the anti-70 kDa and anti-40 kDa antibodies solution were prepared in 10 mmol l−1 phosphate buffer, pH 7·5. 0·5 ml of diluted antibody was mixed with 2·5 ml of gold solution, and the colour of the solution was noted. The minimum amount of the immunoglobulin necessary to stabilize the red colour solution was noted.
One hundred microlitre (2 μg μl−1) of anti-70 kDa and anti-40 kDa antibodies was mixed with 2·5 ml of gold solution separately and incubated for 30 min at room temperature. The mixture was centrifuged at 7000 g for 20 min at 4°C. Supernatant was discarded, and antibody-gold pellet was re-suspended in 500 μl of stabilizing buffer (0·15 mol l−1 NaCl, 10 mmol l−1 Tris-Cl, pH 7·5 0·048% polyethylene glycol (4000) and 0·001% NaN3) and stored at 4°C.
1·0 ml of growing culture of T. fusca was centrifuged at 20 000 rpm for 10 min at 4°C. Supernatant was discarded, and the cell pellet was suspended in 100 μl of 1× PBS. A drop of suspended culture was placed on albumin-coated slide, and a thin smear was made on it. After air drying, one drop of 1% glutaraldehyde in 1× PBS was added on it and kept at room temperature for 8–10 min. After washing, a drop of 3% BSA in PBS was added on it and again kept for 25 min at room temperature. 100 μl of diluted gold-conjugated anti-70 kDa and anti-40 kDa antibodies (1 : 50 dilution) was added on it separately and kept in humidified chamber for 30 min. The slides were washed with PBS, and 1·0% buffered glycerol was added on smear. The coverslip was placed on it and observed under microscope.
Purified S-layers proteins (70 and 40 kDa) of T. fusca were immobilized on charged polystyrene microwell plate (Corning) as follows. 100 μl of S-layer proteins (70 and 40 kDa, total 10 μg in 0·05 mol l−1 carbonate buffer, pH 9·0) was added in microwell plate separately and incubated for 16 h at 4°C. The unbound protein solution was removed, and its concentration was measured by Bradford reagent assay as described above. 100 μl of fixing solution (2% glutaraldehyde in 0·05 mol l−1 carbonate buffer, pH 9·0) was added in each well and incubated for 2 h at 4°C. Wells were washed with 0·05 mol l−1 phosphate buffer, pH 7·4 and used for conjugation of anti-SEB (staphylococcus enterotoxin B antigen) antibodies as follows. 50 ml of 1-ethyl-3(3-dimethylaminopropyl) carbodiimide solution (500 mg ml−1 in 0·05 mol l−1 phosphate buffer, pH 7·4) was mixed with 50 μl of purified anti-SEB antibodies (10 μg in 0·05 mol l−1 phosphate buffer, pH 7·4) and added in wells containing fixed 70- and 40-kDa S-layer proteins. The wells were kept for 4 h at 25°C with mild shaking. The remaining unbound antibody solution was removed, and its concentration was determined by Bradford reagent as described above. The wells were washed with 0·05 mol l−1 phosphate buffer, pH 7·4 and stored at −20°C till further use. The matrixes were designated as 70-SLIM (70-kDa S-layer immunomatrix) and 40-SLIM (40-kDa S-layer immunomatrix).
Purified S. aureus enterotoxin B (SEB toxin) was obtained from Dr. Zahoor Samra Lab (IBB, University of the Punjab). 100 μl of purified SEB toxin (0·5 μg μl−1) and 100 μl of milk contaminated with Staphylococcus aureus were separately added in wells immobilized with 70-SLIM and 40-SLIM. The wells were kept for 45 min at room temperature. Unbound pure SEB toxin was removed and its concentration was determined. SEB toxin adsorbed on 70-SLIM and 40-SLIM was eluted as follows. 100 μl of 0·1 mol l−1 glycine buffer pH 2·5 was added in each well and kept for 30 min at room temperature. The glycine buffer from each well was removed, equilibrated with 0·05 mol l−1 phosphate buffer, pH 7·4 and utilized for the determination of eluted protein by Bradford reagent as described above. Eluted SEB toxins were further characterized by indirect ELISA and 10% SDS-PAGE.
The glycine eluted fractions were also tested to check the leaching of 70-SLIM and 40-SLIM from the wells. 50 μl of glycine-eluted fractions was mixed separately with 50 μl of 0·05 mol l−1 carbonate buffer, pH 9·0 and added in microwell. The microwells were kept at 37°C for 45 min. After washing with TBS, wells were blocked with 3% BSA in TBS for 45 min at 37°C. Again after washing with TBS, 100 μl of rabbit anti-mouse IgG-alkaline phosphatase-conjugated antibodies (1 : 2000 dilution) was added in wells and incubated for 30 min at 37°C. The colour reaction was developed by using para-nitrophenyl phosphate as substrate, and results were noted.
S-layers proteins of Thermobifida fusca were extracted by treatment of whole cells with 4M guanidine hydrochloride. Two major protein bands of 70 and 40 kDa were observed on 10% SDS-PAGE. Extracted protein fractions were loaded on DEAE-cellulose resin and eluted by stepwise gradient. It was observed that 70 and 40 kDa protein fractions were eluted at 0·3 mol l−1 NaCl concentration along with minor fraction of nearly 14 kDa protein. The protein fractions collected from ion exchanger were further fractionated on gel filtration chromatography, and two separate sharp peaks of 70 and 40 kDa were eluted. Protein fractions collected during purification steps were analysed on 10% SDS-PAGE (Fig. 1).
Indirect ELISA was used to confirm the presence of anti-70 kDa and anti-40 kDa in immunized sera. Western blot was performed to determine the monospecificity of anti-70 kDa and anti-40 kDa antibodies towards 70 and 40 kDa S-layer proteins. When anti-70 kDa and anti-40 kDa antibodies were tested to check the cross-reactivity with 70 and 40 kDa proteins, no cross-reactivity was observed (Fig. 2), but both antibodies showed reactivity with total cell lysate. A minor band of 14 kDa was not immunochemically stained with both antibodies.
Immunogold labelling was performed to localize the 70 and 40 kDa S-layer proteins on the surface of T. fusca. The results revealed that 70 and 40 kDa proteins are located on the outer surface of T. fusca (Fig. 3). The direct binding of SEB toxin with S-layer was not detected in ELISA.
S-layer proteins were immobilized onto microwell as a nanomatrix. It is observed that 6·8 μg of 70 kDa proteins and 7·2 μg of 40 kDa protein were bound with wells separately. The conjugation of anti-SEB toxin antibodies was observed at 1·22 μg per 1·0 μg (83·30%) and 0·875 μg μg−1 (63·30%) onto immobilized 70- and 40-kDa S-layer matrixes in wells, respectively. When the wells were used for the binding and elution of pure SEB toxin, it was observed that 5–7 μg of pure SEB toxin was bound in well-contained 70-SLIM and 3–4 μg in well-contained 40-SLIM. It was concluded that more binding capacity of pure SEB toxin was observed onto 70-SLIM. The proteins were also eluted from 70-SLIM and 40-SLIM which were incubated with SEB contaminated milk separately. The eluted proteins were tested on 10% SDS-PAGE, and it was observed that SEB toxin was eluted in free as well as in combined form. 70 and 100 kDa protein bands were observed in samples eluted from 70-SLIM and 60 kDa protein band that was observed in sample eluted from 40-SLIM. The other minor bands of nearly 12 and 20 kDa were also observed (Fig. 4). The eluted fractions were also tested to check the leaching of 70-SLIM and 40-SLIM by indirect ELISA. The absence of substrate colour in ELISA further indicated that the 70-SLIM and 40-SLIM were not leached out from the wells. The proposed model of S-layer matrix is shown (Fig. 5).
S-layers are monomolecular crystalline arrays of proteinaceous subunits decorated as surface structures on archaea and bacteria (Sleytr 1978; Sleytr et al. 1993, 1999). Different methods have been developed for the detachment of S-layer proteins and for their disintegration into protomeric subunits (James et al. 1988; Koval 1988; Messner and Sleytr 1992; Beveridge 1994; Sleytr et al. 1996). It is recommended that most of the S-layer proteins can be solubilized with high concentration of guanidine hydrochloride. The blend of biotechnology and chemistry is opening up many innovative opportunities for the development of bottom-up strategies. The bottom-up informations about S-layer proteins will lead to spectrum applications in biotechnological sciences.
In this study, S-layer proteins (70 and 40 kDa) of Thermobifida fusca was purified, characterized and used as matrix for the development of detection assays. Another minor protein band of nearly 14 kDa was also present. This fraction was not used to develop the SLIM assays. Western blot indicated that this protein fraction was not the intact part of major 70- and 40- kDa proteins. S-layer proteins were immobilized onto microtitre wells and conjugated with anti- Staphylococcus aureus (SEB) toxin antibodies to remove the SEB toxin from the samples. The elution of pure SEB toxin was 1·2 times higher from 70-SLIM than 40-SLIM. Good binding and elution efficiency was observed onto 70-SLIM which may be due to large surface area available for protein to protein interaction. ELISA and Western blot revealed that the antibodies were monospecific towards 70 and 40 kDa S-layer proteins. In immunoassays, the direct binding of SEB toxin with S-layer was not observed. In immunomicroscopic study, the deposition of immunogold particles on the surface of T. fusca cells revealed that the extracted S-layer proteins are the surface proteins of the cells. It also advocated that the method adopted in this study for purifying the S-layers of T. fusca is quite reliable and did not effect on its biochemical specificity, structural stability and reactivity. It is observed that the purification procedure described in this study did not change the self-assembled binding properties of the S-layer proteins on solid surface. ELISA further indicated that SLIMs were not leached out from the wells. It was not tested, either it is monolayer or multilayer. However, most of the S-layer proteins follow monolayer formation during self-assembly process. The self-assembled properties of outer and inner surface showed that it has anisotropic structure. The supramolecular structure of S-layer is being used in different life sciences experiments. The S-layer lattice structure is getting attention for application in nonlife sciences research also. The release of S-layer proteins in high concentration and free from other cellular components is the requirement during isolation of envelope fractions (Boot et al. 1993). It is reported that 45 kDa S-layer of Lactobacillus acidophilus ATCC 4345 was isolated by treating the cells with 4·0 mol l−1 guanidine hydrochloride. Akeokka et al. (1991) isolated 32 and 45 kDa S-layer proteins of Clostridium dificile GAI 0714 by treating the cells with 8 mol l−1 urea in buffer.
Nowadays, the key challenges in biomaterial sciences are the use of reliable self-assembled molecules that can spontaneously associate into supramolecular structures by noncovalent forces. The crystalline bacterial S-layers can self-assemble on lipid contents and represent as a good model for lipoprotein S-layer complex which enhances the long-term stability and increases binding efficiency. S-layer fusion proteins have been constructed by incorporating with IgG, streptavidin, allergens and green fluorescent proteins (Schuster et al. 1998, 2005, 2006).
Drug delivery via nanoparticles is also the main challenge of nanobiotechnology. Liposomes nanoparticles have the advantages of being small, flexible, biocompatible and can pass through smallest arterioles and endothelial fenestrations without causing clotting or damage (Jong and Borm 2008). S-layer-based liposomes nanoparticles may be utilized as an efficient nanocarrier or matrix. Mader et al. (1999) isolated the crystalline S-layer protein of Bacillus stearothermophilus and observed that these can be recrystallized easily on positively charged unilamellar liposomes. Similarly, Seta et al. (1999) showed that the S-layer protein of Bacillus coagulans can be recrystallized on liposomes and cross-linked with glutaraldehyde for making a matrix for the covalent attachment of macromolecules. The stability of S-layer-coated liposomes and promising for immobilizing biomolecules on the crystalline array may have good potential in various liposome applications. Hollmann et al. (2010) demonstrated the possible therapeutic applications of lactobacillar S-layers in the targeted antigen delivery vehicles to the host tissues and suggested that these can be consider as new, safe and stable particles for drug delivery. Due to unique self-assembled binding property of S-layer protein, these can be used for many biochemical process such as (i) preparation of base of affinity matrix, (ii) for the development of immunoassays, (iii) to remove the analytes or toxins from environmental and biological samples through binding of ligands, (iv) immobilization of enzymes, (v) construction of biosensors, (vi) uses as carrier for vaccination to elicit the innate immune response.
Other methods are also available to detect the SEB toxin in biological fluids as well as in environmental samples. These immunomatrixes are not the replacement of other methods, but can be used as an alternate to detect and remove the SEB toxin. The immunomatrixes can be decorated as self-assembled S-layer on solid support. Due to the best scaffold and corrugate property of S-layer, the modern nonlife sciences concur its importance. Due to high temperature tolerance of T. fusca and providing good support, it can be used to immobilize industrially important enzymes.
Thermally stable S-layers of T. fusca can also be used to develop different nanomatrix to remove the toxins or nonrequired biomolecules from the samples. The self-assembled property of S-layer of T. fusca on artificial surfaces such as silicon wafers, noble metals and plastics needs to be tested. The heterologous expression of S-layers of T. fusca is underway for modification to use as building blocks or templates for generating functional nanostructures at nanoscale for diagnostic study.