Generation of a Functional Monomolecular Protein Lattice Consisting

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Bioconjugate Chem. 2003, 14, 440−448

Generation of a Functional Monomolecular Protein Lattice Consisting of an S-Layer Fusion Protein Comprising the Variable Domain of a Camel Heavy Chain Antibody Magdalena Pleschberger,†,‡ Angela Neubauer,‡ Eva M. Egelseer,†,‡ Stefan Weigert,‡ Brigitte Lindner,‡ Uwe B. Sleytr,† Serge Muyldermans,§ and Margit Sa´ra*,†,‡ BMT-Biomolecular Therapeutics GmbH, Brunnerstrasse 59, A-1235 Vienna, Austria, Center for Ultrastructure Research and Ludwig Boltzmann-Institute for Molecular Nanotechnology, University of Agricultural Sciences Vienna, A-1180 Vienna, Austria, and Vrije Universiteit Brussel, Vlaams Interuniversitair Instituut voor Biotechnologie, Paardenstraat 65, B-1640 Sint Genesius Rode, Belgium. Received September 9, 2002; Revised Manuscript Received December 18, 2002

Crystalline bacterial cell surface layer (S-layer) proteins are composed of a single protein or glycoprotein species. Isolated S-layer subunits frequently recrystallize into monomolecular protein lattices on various types of solid supports. For generating a functional protein lattice, a chimeric protein was constructed, which comprised the secondary cell wall polymer-binding region and the self-assembly domain of the S-layer protein SbpA from Bacillus sphaericus CCM 2177, and a single variable region of a heavy chain camel antibody (cAb-Lys3) recognizing lysozyme as antigen. For construction of the S-layer fusion protein, the 3′-end of the sequence encoding the C-terminally truncated form rSbpA31-1068 was fused via a short linker to the 5′-end of the sequence encoding cAb-Lys3. The functionality of the fused cAb-Lys3 in the S-layer fusion protein was proved by surface plasmon resonance measurements. Dot blot assays revealed that the accessibility of the fused functional sequence for the antigen was independent of the use of soluble or assembled S-layer fusion protein. Recrystallization of the S-layer fusion protein into the square lattice structure was observed on peptidoglycan-containing sacculi of B. sphaericus CCM 2177, on polystyrene or on gold chips precoated with thiolated secondary cell wall polymer, which is the natural anchoring molecule for the S-layer protein in the bacterial cell wall. Thereby, the fused cAb-Lys3 remained located on the outer S-layer surface and accessible for lysozyme binding. Together with solid supports precoated with secondary cell wall polymers, S-layer fusion proteins comprising rSbpA31-1068 and cAbs directed against various antigens shall be exploited for building up monomolecular functional protein lattices as required for applications in nanobiotechnology.

INTRODUCTION

Crystalline bacterial cell surface layers (S-layers) represent the outermost cell envelope component of many bacteria and archaea. S-Layer lattices completely cover the cell surface during all stages of bacterial growth and division, and they are mostly composed of a single protein or glycoprotein species. S-Layers exhibit either oblique, square, or hexagonal lattice symmetry (1-3). Depending on the type of S-layer protein, the subunits show a molecular mass of 40 000 to 200 000 Da. In bacteria, the S-layer subunits are linked to each other and to the underlying cell envelope layer by noncovalent interactions. In the case of Bacillaceae, the N-terminal part is involved in anchoring the S-layer subunits to the rigid cell wall layer via a distinct type of glycan, termed secondary cell wall polymer (1, 4-8). Isolated S-layer subunits frequently possess the ability to recrystallize in suspension. This process leads to selfassembly products, which can have the shape of flat sheets or open-ended cylinders (2). The S-layer subunits * To whom correspondence should be addressed: University of Agricultural Sciences Vienna, Gregor Mendelstrasse 33, A-1180 Vienna, Austria. Tel.: +43-1-47 654 2208. Fax: +431-47 89 112. e-mail: [email protected]. † BMT-Biomolecular Therapeutics GmbH. ‡ University of Agricultural Sciences Vienna. § Vrije Universiteit Brussel.

may also recrystallize into monomolecular protein lattices on solid supports, such as gold chips, silicon wafers, or plastics, at the air/water interface, on lipid films or liposomes. The properties of the underlying material allow prediction of the orientation of the S-layer subunits that attach either with their outer or inner surface (3, 9-11). The cell surface of Bacillus sphaericus CCM 2177 is completely covered with a square S-layer lattice, which is composed of identical subunits with a molecular mass of 127 000 Da. The gene encoding this S-layer protein, termed SbpA, had been sequenced, cloned in plasmid pET28a and expressed in Eschericia coli HMS174(DE3) (12). The protein precursor includes a 30 amino acid long typical Gram-positive signal sequence peptide and consists of a total of 1 268 amino acids. Studies on the structure-function relationship of SbpA revealed that 200 C-terminal amino acids could be deleted without interfering with the self-assembly process or the formation of the square lattice structure (12). Furthermore, amino acid position 1 068 was found to be located on the outer S-layer surface and was therefore exploited as fusion site for the production of a chimeric S-layer protein comprising the sequence of the major birch pollen allergen (12). The S-layer protein SbpA recognizes a distinct type of secondary cell wall polymer as the proper anchoring structure in the rigid cell wall layer (13). The polymer chains, which are covalently linked to the

10.1021/bc025603+ CCC: $25.00 © 2003 American Chemical Society Published on Web 02/12/2003

S-Layer Fusion Proteins

peptidoglycan, consist of 8 to 9 disaccharide repeating units with the structure f3)-[4,6-O-(1-carboxyethylidene)]-0.5-β-D-ManpNAc-(1f4)-β-D-GlcpNAc-(1f. The formation of self-assembly products by SbpA or rSbpA strongly depends on the presence of bivalent cations (13). Camel antibodies are part of the humoral immune response of camels and llamas. One group of antibodies of Camelidae are heavy chain dimers, in which the light chains are totally absent. This unique antibody species interacts with the antigen by virtue of a single variable domain, termed VHH (variable domain of a heavy chain of a camel heavy chain antibody). A single VHH domain has a molecular mass of only 15 000 Da and is the smallest known complete antigen binding fragment from a functional immunoglobulin. Despite the absence of the combinatorial diversity of conventional heterodimeric antibodies caused by the VH (variable domain of the heavy chain)-VL (variable domain of the light chain) parts, these heavy chain antibodies exhibit a broad antigen binding repertoire by having enlarged their variable region CDR3 (14, 15). Recombinant VHHs that were selected from VHH libraries are well expressed in E. coli, highly soluble in aqueous environments, and very resistant against denaturation and display high affinity and specificity toward their antigens. Because of their smaller size, VHHs can interact with epitopes that are not recognized by conventional antibody fragments. Due to these properties, VHHs clearly offer an improvement over conventional, more complex, antibody fragments, e. g., in diagnostic applications or as immunoadsorbents, where the stability of the biomolecular probes is critical. Moreover, VHHs constitute ideal modular building blocks for manifold molecular constructs (14, 15). To explore the potential advantages of camel single domain antibodies and to gain insight into how they recognize their target, Desmyter et al. (16) solved the crystal structure of a camel VHH complexed with hen’s egg lysozyme. Lysozyme was chosen as immunogen, because its 3-D structure is well-known and complexes with various antibody fragments have been analyzed before (17-24). Two VHHs that were cloned and expressed in E. coli (25, 26) bound lysozyme in a 1 to 1 stoichiometry with affinity constants of 5 × 10-8 M and 5 × 10-7 M, respectively. These VHHs were termed cAb-Lys2 and cAb-Lys3. In the present study, an S-layer fusion protein comprising the sequence of cAb-Lys3 was constructed. For this purpose, the 3′-end of the gene encoding the Cterminally truncated form of the S-layer protein SbpA, termed rSbpA31-1068, was fused via a short linker to the 5′-end of the gene encoding the VHH fragment of the camel antibody cAb-Lys3 directed against lysozyme. The obtained S-layer fusion protein (theoretical molecular mass of 123 947 Da) represents a model system for the construction of further chimeric proteins, that comprise rSbpA31-1068 and cAbs for generating monomolecular functional protein lattices on solid supports, as required for many applications in nanobiotechnology including biochip development. MATERIAL AND METHODS

Cloning of the Genes Encoding rSbpA31-1068/cAbLys3. All PCR reactions were performed as described in (27). For amplification of the gene encoding rSbpA31-1068, chromosomal DNA of B. sphaericus CCM 2177 was used as template. The oligonucleotide primers sbpA/NcoI [5′CG GAT TCC ATG GCG CAA GTA AAC GAC TAT AAC AAA ATC-3′], which introduced the restriction site (bold)

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NcoI, and the reverse primer sbpA/NheI [5′-GAC CGC GCT AGC TTC TGA ATA TGC AGT AGT TGC TGC C-3′], which introduced the NheI restriction site at the 3′-end, were constructed. The cAb-Lys3 gene was amplified from plasmid cAb-Lys3-pHen6 (16). For this purpose, the oligonucleotide primers cAb/NheI [5′-CGG ATT GCT AGC GAT GTG CAG CTG CAG GCG-3′] and cAb/XhoI [5′-GAC CGC TCG AGT TAT GAG GAG ACG GTG ACC TGG G-3′], which introduced the restriction sites NheI and XhoI, were used. Because of the GC-rich sequence of cAb-Lys3, betaine was added to a final concentration of 1.5 M to the PCR mixture. To obtain the plasmid pET28a-sbpA(93-3204), the gel-purified PCR product sbpA(93-3204), encoding the truncated form of the S-layer protein, was ligated into the corresponding restriction sites of plasmid pET28a, which was established in E. coli TG1. For generating the chimeric sbpA(93-3204)/cAb-Lys3 gene, the gel-purified PCR product cAb-Lys3, encoding the VHH domain of the camel antibody directed against lysozyme, was ligated into the corresponding restriction sites of plasmid pET28a-sbpA(93-3204), which was used for transformation of E. coli TG1. Expression of the Chimeric sbpA(93-3204)/cAb-Lys3 Gene. The plasmid stability test and heterologous expression of the sbpA(93-3204)/cAb-Lys3 gene in E. coli HMS174(DE3) were performed as described in (27). Samples of the E. coli HMS174(DE3) cultures were taken 1 to 5 h after induction of sbpA(93-3204)/cAb-Lys3 gene expression by the addition of 1 mM isopropyl β-Dthiogalactopyranoside (IPTG, GEBRU). Preparation of samples for SDS-PAGE analysis was carried out as described by Laemmli (28). For electron microscopic investigation, whole cells of E. coli HMS174(DE3) were prepared as described in ref 29. Electron microscopic investigations were done with a Philips CM 100 transmission electron microscope (TEM). Isolation of the S-Layer Fusion Protein from the Host Cells and Purification. Isolation of rSbpA31-1068/ cAb-Lys3 from E. coli HMS174(DE3) was performed as described in a previous study (30). After lysis of the host cells, 100 mg wet pellet, obtained by centrifugation at 30 000g for 15 min at 4 °C, was suspended in 4 mL of 5 M guanidine hydrochloride in 50 mM Tris-HCl buffer (pH 7.2). Then the suspension was centrifuged at 36 000g for 30 min at 4 °C, the supernatant containing the S-layer fusion protein was diluted 1:2.5 with 50 mM Tris-HCl buffer (pH 7.2), and the clear solution was subjected to gel permeation chromatography using a Superdex 200 column (Pharmacia) equilibrated in 2 M guanidine hydrochloride in 50 mM Tris-HCl buffer (pH 7.2). Fractions belonging to the first peak were pooled and dialyzed against MilliQ-water for 18 h at 4 °C, lyophilized, and stored at -20 °C. All guanidine hydrochloride solutions and buffers used during the isolation and purification procedure contained 150 mM NaCl. Immunoblotting using a polyclonal rabbit antiserum raised against the S-layer protein of B. sphaericus CCM 2177 was performed as described (31). The presence of the cAb-Lys3 moiety in the S-layer fusion protein was checked by immunoreactivity with a polyclonal rabbit anti-camel antiserum (Sigma C7540). Investigation of the Self-Assembly Properties of the S-Layer Fusion Protein and Recrystallization on Peptidoglycan-Containing Sacculi. Two milligrams of the gel permeation chromatography-purified and lyophilized S-layer fusion protein were dissolved in 1 mL 5 M guanidine hydrochloride in 50 mM Tris-HCl buffer (pH 7.2) and the solution was dialyzed against 10 mM CaCl2 in MilliQ-water containing 1 mM 1,4-dithio-

442 Bioconjugate Chem., Vol. 14, No. 2, 2003 DL-threitol (DTT) for 18 h at 4 °C. For recrystallization of the S-layer fusion protein on peptidoglycan-containing sacculi, 2 mg rSbpA31-1068/cAb-Lys3 was dissolved in 1 mL of 5 M guanidine hydrochloride in 50 mM Tris-HCl buffer (pH 7.2) and 2 mg of lyophilized peptidoglycancontaining sacculi of B. sphaericus CCM 2177, which was prepared according to (12, 13), was added. The suspension was dialyzed under conditions described above. Peptidoglycan-containing sacculi carrying a monolayer of recrystallized S-layer fusion protein are referred to as recrystallization products in all further experiments. To keep the S-layer fusion protein water soluble, the guanidine hydrochloride-extract was dialyzed against 1 mM DTT in MilliQ-water. Localization of the Fused cAb-Lys3 by Immunogold-Labeling of Self-Assembly and Recrystallization Products. Self-assembly and recrystallization products obtained with rSbpA31-1068/cAb-Lys3 were incubated with a polyclonal rabbit antiserum raised against camel antibodies [Sigma C7540; diluted 1:2 with 50 mM Tris-HCl buffer (pH 7.2)] at 20 °C for 1 h. After centrifugation of the suspensions at 16 000 × g for 20 min at 4 °C and two washing steps with TBS (50 mM Tris-HCl, pH 7.2, containing 0.15 M NaCl), the pellets were resuspended in 100 µL solution of a Protein A-colloidal gold conjugate (10 nm, Sigma P1039) and incubated at 20 °C for 2 h. All further steps were carried out as described in ref 12. Surface Plasmon Resonance (SPR) Studies for Investigation of the Functionality of the Fused cAb-Lys3. SPR experiments were performed with a Biacore 2000 system (BIACORE, Sweden). Lysozyme was immobilized on a CM5 chip on flow cell 1 (FC1). For this purpose the carboxylic acid groups on the sensor chip were activated by injecting 35 µL of a solution of 400 mM 1-ethyl-3-(3-diaminopropyl)carbodiimide (EDC; Sigma E7750) and 100 mM N-hydroxysuccinimide in MilliQwater. Thereafter, 50 µL of a solution of 0.5 mg/mL lysozyme (Sigma L6876) in MilliQ-water was injected. The immobilization procedure was completed by blocking with 35 µL of 1 M ethanolamine hydrochloride, dissolved in MilliQ-water at pH 8.5. Continuous flow buffer was HBS (10 mM HEPES, pH 7.2, containing 0.15 M NaCl and 0.005% Tween 20). The amount of immobilized lysozyme was expressed in terms of resonance units (RU; 1000 RU correspond to approximately 1 ng/mm2). Untreated carboxymethylated dextran on flow cell 2 (FC2) was used as reference surface. For interaction studies, gel permeation-chromatography purified, lyophilized rSbpA and rSbpA31-1068/cAb-Lys3 were dissolved in HBS buffer at a concentration of 0.8 µmol/mL. During the reaction time, a continuous flow of HBS buffer (10 µL/ min) was maintained over the sensor surface. The contact and dissociation time of rSbpA31-1068/cAb-Lys3 with the surface was 20 min and 800 s, respectively. Dot Blot Assays for Investigating the Accessibility of the Fused cAb-Lys3. To investigate the accessibility of the fused cAb-Lys3 in the water soluble, selfassembled, or recrystallized S-layer fusion protein, 5 µL samples with an S-layer protein content of 1 mg/mL were dried onto a nitrocellulose membrane. After blocking with 2% Top Block (Fluka) in TBS and incubating in 20 mL of lysozyme solution (250 µg/mL TBS containing 2% Top Block) for 1 h at 20 °C, the membrane was washed three times with washing buffer (TBS containing 0.5% Tween) and finally incubated in a polyclonal rabbit antiserum raised against lysozyme (Rockland 200-4172; diluted 1:4000 in washing buffer containing 2% Top Block). After further washing, the membrane was incubated with a

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solution of anti-rabbit IgG alkaline phosphatase-conjugate (Sigma A3687; diluted 1:5000 in blocking solution) for 1 h at 20 °C. Bound antibody was finally visualized by using BCIP/NBT (Roche 1681451) as substrate for the alkaline phosphatase. Recrystallization of the S-Layer Fusion Protein on Polystyrene and Comparison of the Ability of the Formed Monolayer To Bind Lysozyme with That of a Monolayer Generated by Recrystallization of rSbpA and Chemical Coupling of cAb-Lys3 by Means of ELISA. Lyophilized S-layer fusion protein (2 mg) was dissolved in 1.2 mL of 5 M guanidine hydrochloride in 50 mM Tris-HCl buffer (pH 7.2) and dialyzed against 1 mM DTT in MilliQ-water for 18 h at 4 °C. After centrifugation at 30 000g for 15 min at 4 °C, the clear supernatant was diluted to a final protein concentration of 100 µg/mL with 50 mM Tris-HCl buffer (pH 9.0) containing 10 mM CaCl2. The wells of a polystyrene ELISA plate (Greiner Austria, 665101) were filled with 300 µL aliquots of the protein solution and incubated for 18 h at 4 °C for recrystallization. After rinsing with MilliQ-water, samples were taken for AFM analysis. Recrystallization of rSbpA was performed under the same conditions. After cross-linking the S-layer lattice with glutaraldehyde (1% in TBS), activation of free carboxylic acid groups with EDC (32), and washing with ice cold MilliQ-water, 100 µL of a cAb-Lys3 solution (10 µg/mL MilliQ-water, pH adjusted to 9.0 with 0.1 M NaOH) was added and incubated for 18 h at 4 °C. Unbound protein was removed by washing with TBS containing 0.1% TRITON X-100. To investigate the ability to bind lysozyme, both types of monolayers were incubated with various concentrations of biotinylated lysozyme (3, 6, or 12 ng in TBS containing 1% TRITON X-100 per well) for 1 h at 20 °C. After rinsing with MilliQ-water and TBS containing 1% TRITON X-100, 100 µL of a streptavidin-peroxidase conjugate (Amersham RPN 4401; diluted 1:5 000 in TBS containing 1% TRITON X-100) was transferred into the wells, and the plates were incubated for 1 h at 20 °C. Bound streptavidin-peroxidase conjugate was visualized by using 3,3′,5,5′-tetramethylbenzidine (TMB; Sigma T3405) as substrate for peroxidase. Absorption was measured at 450 nm in an ELISA reader (E-Liza Mat3000, DRG). As a reference, the ELISA was performed on plain rSbpA. For determination of the amount of cAb-Lys3 that could be covalently bound to the rSbpA monolayer, the S-layer protein was recrystallized on polystyrene beads with a diameter of 1 µm (Bangs Laboratories PC03N) under the same conditions as described for the ELISA wells. After cross-linking the S-layer lattice with glutaraldehyde and activation of free carboxylic acid groups with carbodiimide, cAb-Lys3 was immobilized under conditions described above. The amount of covalently bound cAb-Lys3 was determined as described in previous studies (32-34). The presence of the square lattice structure was checked by freeze-etching of the polystyrene beads and TEM analysis (29). Binding of Thiolated Secondary Cell Wall Polymer to Plain Gold Chips and Recrystallization of rSbpA and the S-Layer Fusion Protein. Secondary cell wall polymer was isolated from peptidoglycancontaining sacculi of B. sphaericus CCM 2177 and purified (13). Chemical modification of the reducing end on the polymer chains and introduction of a terminal sulfhydryl group by modification with 2-iminothiolane was performed (35). Gold chips (silicon 〈100〉, coated with 1

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nm Ti and 60 nm Au) with an area of 1 cm2 were cleaned by incubation in a solution containing 50 mM KOH and 15% H2O2 for 30 min at 60 °C, extensive rinsing with MilliQ-water and drying in an oven at 150 °C for 1 h. The clean gold chips were incubated in a solution containing 100 ng of thiolated secondary cell wall polymer in 1 mL MilliQ-water (pH adjusted to 3.5 with 10 mM HCl) at 20 °C for 1 h and washed five times with MilliQwater. Recrystallization of rSbpA and rSbpA31-1068/cAbLys3 on plain gold chips or on gold chips precoated with thiolated secondary cell wall polymer was performed under the same conditions as described for polystyrene. Atomic Force Microscope (AFM) Analysis. A Nanoscope III AFM (Digital Instruments, Santa Barbara, CA) equipped with a liquid cell and a 120 µm scanner and oxide-sharpened Si3N4 tips (Nanoprobes, Digital Instruments, Santa Barbara, CA) with a nominal spring constant of 0.06 N/m was used for all AFM investigations. Imaging of rSbpA or rSbpA31-1068/cAb-Lys3 after recrystallization on solid supports was carried out in contact mode under liquid (100 mM NaCl). Height and deflection signals were recorded simultaneously (error signal mode). The applied force was kept at about 1 nN and the scan rate was 5.1 Hz at a scan size of 1 by 1 µm. Particular care was taken to avoid dewetting of the samples when mounting them in the fluid cell. Force versus distance curves on plain gold chips or gold chips precoated with thiolated secondary cell wall polymer were recorded at 1 Hz. Measurements were carried out in the following electrolytes: 1 and 100 mM NaCl in 1 mM glycine-HCl buffer (pH 3.5), 1 mM Tris-HCl buffer (pH 7.2), or 1 mM Tris-HCl buffer (pH 9.0). Evaluation of force data was carried out using SPIP software (Image Metrology, Lyngby, Denmark). RESULTS

Cloning and Expression of the Gene Encoding rSbpA31-1068/cAb-Lys3. The PCR product which was obtained by amplification of the sbpA gene using the primers sbpA/NcoI and sbpA/NheI and which encoded the truncated form of the S-layer protein SbpA (rSbpA31-1068) was ligated into the pET28a vector. After cloning in E. coli TG1, amplification, and isolation of the plasmid pET28a-sbpA(93-3204), the gene encoding a single VHH domain of a camel antibody recognizing lysozyme, termed cAb-Lys3, was ligated via the corresponding restriction sites into this plasmid. The resulting vector pET28asbpA(93-3204)/cAb-Lys3 was established in E. coli HMS174(DE3). After induction of expression by the addition of IPTG, biomass samples of E. coli HMS174(DE3) were harvested at various points of time and subjected to SDS-PAGE analysis and ultrathin sectioning. In comparison to E. coli HMS174(DE3) cells harvested before the addition of IPTG (Figure 1; lane 1), an additional high molecular mass protein band was observed on SDS-gels in samples from E. coli HMS174(DE3) cultures induced to express the chimeric sbpA(93-3204)/cAb-Lys3 gene (Figure 1; lanes 2 and 3). This additional protein band had an apparent molecular mass of 124 000 Da. Isolation, Purification, and Characterization of the S-Layer Fusion Protein. As derived from SDSPAGE analysis, the recombinant S-layer fusion protein had accumulated in the insoluble fraction of the lysed E. coli HMS174(DE3) cells (data not shown). This was in agreement with data from ultrathin-sectioned preparations of whole cells, which revealed the presence of inclusion bodies in the cytoplasm of the host cells, as it was observed in a previous study for rSbpA (12). The

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Figure 1. SDS PAGE analysis patterns (lanes 1-3) of SDSextracts of whole cells of E. coli HMS174(DE3) harboring the pET28a-sbpA(93-3204)/cAb-Lys3 gene construct before (lane 1) and 2 h (lane 2) and 5 h (lane 3) after induction of sbpA(93-3204)/cAbLys3 expression. Lane 4: SDS-extracts of gel permeation chromatography purified rSbpA31-1068/cAb-Lys3. Immunoblot analysis of SDS-extracts of gel permeation chromatography purified rSbpA31-1068/cAb-Lys3 (lanes 5 and 6). The presence of the SbpA-portion was checked with anti-SbpA antiserum and anti-rabbit IgG-alkaline phosphatase conjugate (lane 5). The presence of the cAb-Lys3 moiety was checked with anti-camel antiserum and anti-rabbit IgG-alkaline phosphatase conjugate (lane 6).

insoluble fraction of the lysed E. coli HMS174(DE3) cells was extracted with guanidine hydrochloride and subjected to gel permeation chromatography. After unifying the fractions from the first peak and removing guanidine hydrochloride by dialysis, only a single major protein band with an apparent molecular mass of 124 000 Da was observed on SDS-gels (Figure 1; lane 4). On immunoblots, a strong cross reaction was observed between the high molecular mass protein band and the polyclonal rabbit antiserum raised against the S-layer protein SbpA of B. sphaericus CCM 2177, as well as with the polyclonal rabbit antiserum raised against camel serum (Figure 1; lanes 5 and 6). The identification of the high molecular mass protein band as the S-layer fusion protein rSbpA31-1068/cAb-Lys3 was finally accomplished by Nterminal sequencing. The first five amino acids (AQVND) were identical to those of the S-layer protein SbpA (12). To investigate the self-assembly properties, purified and lyophilized S-layer fusion protein was denatured in 2 M guanidine hydrochloride, and the solution was subsequently dialyzed against 10 mM CaCl2 in 1 mM DTT. As shown by negative-staining, the S-layer fusion protein reassembled into flat sheets, which clearly exhibited the square lattice structure (not shown). Self-assembly products were mostly double layers, but also multilayers were observed. The S-layer fusion protein recognized peptidoglycan-containing sacculi of B. sphaericus CCM 2177 as binding site and recrystallized into the square lattice (Figure 2A). Localization of the Fused cAb-Lys3 by Immunogold-Labeling of Self-Assembly and Recrystallization Products. Self-assembly and recrystallization products obtained with rSbpA31-1068/cAb-Lys3 were labeled with a polyclonal rabbit anti-camel antiserum and bound antibodies were visualized with a Protein A-colloidal gold conjugate. Negatively stained preparations of self-assembly products (not shown) and recrystallization products (Figure 2B) prepared with rSbpA31-1068/cAb-Lys3 were densely labeled with the Protein A-colloidal gold conjugate, while self-assembly and recrystallization products formed by rSbpA remained completely unlabeled (not shown). Because of the specific orientation of the S-layer fusion protein in recrystallization products, the inner S-layer surface was blocked, while the outer face was exposed to the environment. This finally confirmed

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Figure 4. Dot Blots assays indicating the accessibility of cAbLys3 in the water soluble, self-assembled, and recrystallized S-layer fusion protein. Spot 1: rSbpA; spot 2: water soluble rSbpA31-1068/cAb-Lys3; spot 3: self-assembly products formed by rSbpA31-1068/cAb-Lys3; spot 4: recrystallization products obtained with rSbpA31-1068/cAb-Lys3.

Figure 2. Electron micrographs of negatively stained preparations of recrystallization products formed by rSbpA31-1068/cAbLys3, (A) before and (B) after binding of anti-camel antibodies and labeling with a Protein A-colloidal gold conjugate. Bars, 100 nm.

Figure 3. Sensorgram showing the association (s) and dissociation (- - -) between lysozyme immobilized on the CM5 sensor chip surface and water soluble rSbpA31-1068/cAb-Lys3. The sensorgram indicates specific affinity of rSbpA31-1068/cAb-Lys3 to lysozyme.

that the cAb-Lys3-portion was located on the outer surface of the S-layer lattice (Figure 2 B). Surface Plasmon Resonance (SPR) Studies for Investigation of the Functionality of the Fused cAb-Lys3. To investigate the functionality of the fused cAb-Lys3 by means of SPR measurements, a solution of rSbpA31-1068/cAb-Lys3 in HBS buffer was conducted over a CM5 sensor chip on which 900 RU of lysozyme had been covalently bound. In Figure 3, the untreated CM5 sensor surface which was used as a reference surface was subtracted from the sensorgram obtained with rSbpA31-1068/cAb-Lys3. The two separate sensorgrams clearly showed that the water soluble rSbpA31-1068/cAbLys3 exhibited affinity toward lysozyme, but did not bind

to the plain CM5 surface at all. To exclude the possibility of nonspecific interactions between the S-layer protein moiety and lysozyme, a solution of rSbpA was used in an additional experiment. Since rSbpA showed absolutely no affinity to lysozyme (curve not shown), it was confirmed that binding of the S-layer fusion protein had occurred due to specific interactions between the cAbLys3-moiety and the immobilized antigen. Dot Blot Assays for Investigating the Accessibility of the Fused cAb-Lys3. Dot blot assays were performed to investigate the accessibility of the fused cAb-Lys3 in the water soluble, self-assembled, and recrystallized state of the S-layer fusion protein. As shown in Figure 4, the reaction toward lysozyme was comparable for all samples, independent of the state of the S-layer fusion protein. Assuming an identical functionality of cAb-Lys3 in all S-layer fusion protein samples, these findings indicated that the accessibility of the fused functional sequence for the antigen was not reduced in the self-assembled or recrystallized S-layer fusion protein. Recrystallization of the S-Layer Fusion Protein on Polystyrene and Comparison of the Ability of the Formed Monolayer To Bind Lysozyme with That of a Monolayer Generated by Recrystallization of rSbpA and Chemical Coupling of cAb-Lys3 by Means of ELISA. AFM analysis revealed that rSbpA and the S-layer fusion protein were capable of recrystallizing into the square lattice structure on polystyrene, which was the material of the ELISA wells. Both types of S-layer monolayers consisted of numerous randomly orientated monocrystalline patches with an average size of 100 nm (Figure 5A). The patches clearly displayed the square lattice structure with a center to center spacing of the morphological units of 13.1 nm, which is characteristic of the S-layer protein SbpA from B. sphaericus CCM 2177 (12). For quantification of the amount of cAb-Lys3 that could be chemically coupled to the rSbpA monolayer, the S-layer protein was recrystallized on polystyrene beads with a diameter of 1 µm. Freeze-etching revealed that the beads were completely covered with the square S-layer lattice (not shown). On all beads, the smooth outer S-layer surface was exposed, indicating that the subunits had attached with their more corrugated inner surface (9). After cross linking the S-layer protein with glutaraldehyde and activation of free carboxylic acid groups with carbodiimide, 130 ng cAb-Lys3 could be immobilized per cm2 monomolecular S-layer lattice. Considering that the molecular mass of cAb-Lys3 is 15 000 Da, this amount corresponds to 8-9 cAb-Lys3 molecules per morphological unit or to at least 2 cAbLys3 molecules per S-layer subunit. For comparison, only 4 cAb-Lys3 residues were available per morphological unit in the case of the S-layer fusion protein. Despite this difference, both types of monolayers gave comparable

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Figure 5. AFM images of (A) rSbpA recrystallized on polystyrene and (B) rSbpA and (C) rSbpA31-1068/cAb-Lys3 recrystallized on gold chips precoated with thiolated secondary cell wall polymer. In B and C, spherical clusters of ∼50 nm diameter resembling the topography of the sputtered gold surfaces are visible underneath the S-layer lattice. Images were recorded with simultaneous acquisition of the deflection signal and height signal (not shown). Bars, 100 nm.

Figure 6. ELISA curves to compare the ability of the monolayer formed by the S-layer fusion protein (9) and the monolayer generated by recrystallization of rSbpA and chemical coupling of cAb-Lys3 (2) to bind biotinylated lysozyme (concentration in ng/well). rSbpA (b) was used as a blank.

signals in the ELISA to detect lysozyme. No binding of lysozyme was observed on the rSbpA monolayer used as reference substrate (Figure 6). Binding of Thiolated Secondary Cell Wall Polymer to Plain Gold Chips and AFM Analysis. To exploit the specific interactions between the S-layer protein and its natural anchoring molecule in the bacterial cell wall (12, 13) and to favor recrystallization under in vivo like conditions in order to minimize the number of grain boundaries as observed on polystyrene, the gold chips were precoated with thiolated secondary cell wall polymer. Successful binding of the secondary cell wall polymer was confirmed by comparing force versus distance curves measured on plain gold chips with those obtained on gold chips precoated with thiolated secondary cell wall polymer. On clean gold chips, force versus distance curves typically displayed a clear jump-tosurface by van der Waals forces upon approach and some adhesion upon retraction of the tip independent of the ionic strength or pH of the electrolyte (Figure 7a). This was the expected behavior of a Si3N4 tip and a clean, hard, hydrophobic surface in the absence of long-range interactions. In contrast, approach curves recorded on gold chips precoated with thiolated secondary cell wall polymer displayed a smooth, exponentially increasing, long range repulsive force (Figure 7b) with a decay length between 6 and 20 nm depending on the pH and the ionic strength of the electrolyte. A curve morphology like that is either caused by repulsive electrostatic forces (36, 37)

or by polymer brushes grafted to a surface (38, 39). Since in the case of the secondary cell wall polymer net negatively charged carbohydrate chains (13) were bound to the gold surface, force versus distance curves were most likely effected by both of the above-mentioned types of long-range forces. The Si3N4 tip used also had a negative charge at pH > 6 (37, 40). The decay length of the repulsive force observed in the approach curves increased significantly from 6 to 20 nm, when the pH was increased from 3.5 to 7.2, reflecting the increase in charge density on both, the tip and polymer chains at higher pH. At the same time, there was only a moderate dependence of the decay length on the ionic strength. At pH 7.2, this was 20 nm in 1 mM NaCl and 15 nm in 100 mM NaCl. Since the range of electrostatic double layer forces is limited (∼1 nm) in 100 mM NaCl, these findings indicated a significant impact of polymer brush forces to the observed repulsion. Thus it became obvious that under physiological conditions, the carbohydrate chains actually formed the expected structure with the individual chains reaching into the aqueous phase on the gold chip. The lack of adhesion in the retraction curves on gold chips precoated with thiolated secondary cell wall polymer was attributed to the hydrophilic nature of the coated surface. Recrystallization of rSbpA and the S-Layer Fusion Protein on Plain Gold Chips and Gold Chips Precoated with Thiolated Secondary Cell Wall Polymer and AFM Analyses. The recombinant S-layer protein rSbpA was capable of recrystallizing into the square lattice structure on plain gold chips. As shown for polystyrene (Figure 5A), the S-layer monolayer consisted of randomly aligned monocrystalline S-layer patches with a size of approximately 200 nm, which clearly exhibited the square lattice structure. In contrast, a regularly structured protein lattice with significantly improved long range order was generated, when gold chips precoated with thiolated secondary cell wall polymer were used for recrystallization of rSbpA (Figure 5B). The S-layer fusion protein did not recrystallize on plain gold chips at all (not shown), but a monomolecular protein array consisting of extended patches of crystalline domains with square lattice symmetry was formed on gold chips precoated with thiolated secondary cell wall polymer (Figure 5 C). DISCUSSION

Studies on the structure-function relationship of the S-layer protein SbpA of B. sphaericus CCM 2177 were considered as presupposition for the construction of S-layer fusion proteins, which retained the specific self-

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Figure 7. AFM analysis. Force versus distance curves measured on (a) plain gold and on (b) gold chips precoated with thiolated secondary cell wall polymer. The curves shown here were obtained in an electrolyte solution consisting of 100 mM NaCl in 1 mM Tris-HCl buffer (pH 7.2) using oxide-sharpened silicon nitride tips. Curves shown here were selected from the whole set of data collected in different electrolytes (curve morphologies are typical of the respective surfaces). The decay lengths of the repulsive force recorded on gold chips precoated with thiolated secondary cell wall polymer were evaluated after correction of the curves for the additional distance due to cantilever deflection. The approach and retraction parts of the curves are indicated by arrows.

assembly properties of the S-layer protein moiety, as well as the functionality of the fused peptide sequence. These studies revealed that the N-terminal part of SbpA located on the inner S-layer surface was required for anchoring the S-layer subunits via a pyruvylated secondary cell wall polymer to the rigid cell wall layer (13). Furthermore, it could be demonstrated that the deletion of 200 C-terminal amino acids had no influence on the self-assembly properties of this S-layer protein (12). For screening an amino acid position in the C-terminal part that is located on the outer S-layer surface and therefore well-suited for the fusion of functional peptide sequences, the sequence encoding the affinity peptide Strep-tag I was linked to the 3′-end of the sequence encoding rSbpA31-1268 or rSbpA31-1068. Binding of streptavidin to rSbpA31-1268/ Strep-tag I and rSbpA31-1068/Strep-tag I, which were either provided in water soluble form, as self-assembly or recrystallization products, revealed that Strep-tag I was accessible to a significantly higher extent in the C-terminally truncated form. Within the different rSbpA31-1068/Strep-tag I samples, the amount of bound streptavidin was independent of the use of soluble or assembled S-layer protein, which indicated that Streptag I was not located on the contact sites of adjacent S-layer subunits. Since Strep-tag I was found to be exposed on the outer S-layer surface, rSbpA31-1068 was used in a previous study as base form for the construction of an S-layer fusion protein, which carried the sequence of the major birch pollen allergen (Bet v1) at the C-terminal end (12). In the present study, the C-terminally truncated rSbpA form was also selected as base form for the construction of an S-layer fusion protein comprising the sequence of a single variable region of a camel heavy chain antibody directed against lysozyme (cAb-Lys3). The obtained Slayer fusion protein, termed rSbpA31-1068/cAb-Lys3, can be considered as a model system for the construction of further chimeric proteins, which comprise rSbpA31-1068 and cAbs directed against various antigens. The S-layer fusion protein was capable of self-assembling in vitro into flat sheets, which clearly resembled the square lattice structure of the native S-layer. To prove whether the fused cAb-Lys3 sequence was located on the outer S-layer surface, the S-layer fusion protein was recrystallized on peptidoglycan-containing sacculi of B. sphaericus CCM 2177, to which the S-layer subunits attach with their inner surface. Immunogold-labeling

using an anti-camel antiserum and a colloidal goldProtein A conjugate confirmed the location of cAb-Lys3 on the outer S-layer surface. Dot blot assays were performed to compare the accessibility of cAb-Lys3 in the soluble, self-assembled, and recrystallized S-layer fusion protein. According to the data obtained with rSbpA31-1068/ Strep-tag I and streptavidin (12, 41), the accessibility of the fused functional sequence for lysozyme was independent of the soluble or assembled state of the S-layer fusion protein. As shown by AFM analysis, the S-layer fusion protein was capable of recrystallizing into the square lattice structure on polystyrene. The ability of cAb-Lys3 in the formed monolayer to bind lysozyme was investigated and compared with that of a monolayer generated by recrystallization of rSbpA and chemical coupling of cAb-Lys3. Although in the case of the S-layer fusion protein only 4 cAb-Lys3 residues were available per tetrameric morphological unit of the square S-layer lattice, the signal in the assay to detect lysozyme was similar to that for the rSbpA monolayer carrying 8-9 chemically coupled cAb-Lys3 molecules per morphological unit. Since lysozyme has a molecular mass of 14 600 Da, which is comparable to the molecular mass of cAb-Lys3 of ∼15 000 Da, it is not likely that the reduced signal in relation to the number of chemically coupled cAb-Lys3 molecules is due to sterical hindrance of the antigen. An important factor that has to be considered is the procedure applied to detect lysozyme, in which a streptavidin peroxidaseconjugate was used to bind to the biotinylated antigen and where sterical factors may have played a role. Due to the presence of at least 2 chemically coupled cAb-Lys3 molecules per S-layer subunit, the large molecular mass of the streptavidin-peroxidase conjugate, the tetrameric nature of streptavidin and the size of one morphological unit of the S-layer lattice consisting of 4 subunits, it is feasible that a single streptavidin-peroxidase conjugate molecule could bind to two lysozyme molecules. In the case of the S-layer fusion protein, a single cAb-Lys3 residue was available per S-layer subunit, which due to sterical reasons could most probably react with the streptavidin-peroxidase conjugate in a 1 to 1 stoichiometry. This may explain why despite the different numbers of cAb-Lys3 residues available, both types of monolayers gave comparable signals in the assay to detect lysozyme. The cAb-Lys3 sequence was linked to an amino acid position in the S-layer protein, which in a previous study

S-Layer Fusion Proteins

(12) was found to be located on the outer S-layer surface. This should ensure that the fused functional sequence remained accessible for binding of target molecules, provided that the S-layer subunits attached with their inner surface to the supporting layer, which was the case when polystyrene was used as solid support. To achieve oriented binding and recrystallization of the S-layer fusion protein on gold chips, the specific interactions between the S-layer protein and its natural anchoring molecule in the bacterial cell wall, the secondary cell wall polymer (13), were exploited. For this purpose, the latent aldehyde group at the reducing end of the polymer chains was converted into a thiol group (35). By applying this modification procedure, each polymer chain carried a single thiol group, which was capable of binding to the gold surface and guaranteed that the polymer chains attached in uniform orientation. To summarize, S-layer fusion proteins consisting of rSbpA31-1068 and cAbs directed against various antigens shall be exploited for recrystallization on solid supports to generate functional monomolecular protein lattices, as required for applications in nanobiotechnology including biochip development. Due to the selected fusion site, the functional sequence remained located on the outer Slayer surface and was accessible for binding of target molecules, provided that the S-layer subunits attached with their inner surface. To ensure this, secondary cell wall polymers shall be exploited as S-layer specific biomimetic linker to the solid support in future developments. Because of the crystalline structure of the S-layer lattice, the fused functional sequences are arranged at well-defined distance and orientation to each other in the nanometer range. ACKNOWLEDGMENT

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