Biomedical subjects

Eva M Egelseer

Publications and source records attributed to Eva M Egelseer.

12 recordsLinked to original sources

PubMed2006

S-Layers as a basic building block in a molecular construction kit.

Crystalline arrays of protein or glycoprotein subunits forming surface layers (S-layers) are the most common outermost envelope components of prokaryotic organisms (archaea and bacteria). The wealth of information on the structure, chemistry, genetics, morphogenesis, and function of S-layers has revealed a broad application potential. As S-layers are periodic structures, they exhibit identical physicochemical properties for each molecular unit down to the subnanometer level and possess pores of identical size and morphology. Many applications of S-layers in nanobiotechnology depend on the ability of isolated subunits to recrystallize into monomolecular lattices in suspension or on suitable surfaces and interfaces. S-Layer lattices can be exploited as scaffolding and patterning elements for generating more complex supramolecular assemblies and structures, as required for life and nonlife science applications.

Animals↗

PubMed2006

Heterotetramers formed by an S-layer-streptavidin fusion protein and core-streptavidin as a nanoarrayed template for biochip development.

Based on the S-layer protein SbpA of Bacillus sphaericus CCM 2177, an S-layer-streptavidin fusion protein was constructed. After heterologous expression, isolation of the fusion protein, and refolding, functional heterotetramers were obtained that had retained the ability to recrystallize into the square-lattice structure on plain gold chips and on gold chips precoated with secondary cell wall polymer (SCWP), which is the natural anchoring molecule for the S-layer protein in the bacterial cell wall. Monolayers generated by recrystallization of heterotetramers on plain gold chips or on gold chips precoated with thiolated SCWP were exploited for the binding of biotinylated oligonucleotides (30-mers). Hybridization experiments with complementary fluorescently labeled oligonucleotides carrying one mismatch or no mismatch (both 15-mers) were performed and evaluated with surface-plasmon-field-enhanced fluorescence spectroscopy. For surfaces generated by the recrystallization of heterotetramers on SCWP-coated gold chips, a detection limit of 1.57 pM could be determined, whereas for surfaces obtained by direct recrystallization of heterotetramers on plain gold chips, a detection limit of 8.2 pM was found. Measuring the association and dissociation processes of oligonucleotides carrying no mismatch led to a dissociation constant of K(D)=6.3 x 10(-10) m, whereas for oligonucleotides carrying one mismatch a dissociation constant of K(D)=7.9 x 10(-9) m was determined. This finding was confirmed by measuring the whole Langmuir isotherm, which resulted in a dissociation constant of K(D)=2.6 x 10(-8) m.

Biotinylation↗

PubMed2005

The three S-layer-like homology motifs of the S-layer protein SbpA of Bacillus sphaericus CCM 2177 are not sufficient for binding to the pyruvylated secondary cell wall polymer.

The S-layer protein SbpA of Bacillus sphaericus CCM 2177 recognizes a pyruvylated secondary cell wall polymer (SCWP) as anchoring structure to the peptidoglycan-containing layer. Data analysis from surface plasmon resonance (SPR) spectroscopy revealed the existence of three different binding sites with high, medium and low affinity for rSbpA on SCWP immobilized to the sensor chip. The shortest C-terminal truncation with specific affinity to SCWP was rSbpA(31-318). Surprisingly, rSbpA(31-202) comprising the three S-layer-like homology (SLH) motifs did not bind at all. Analysis of the SbpA sequence revealed a 58-amino-acid-long SLH-like motif starting 11 amino acids after the third SLH motif. The importance of this motif for reconstituting the functional SCWP-binding domain was further demonstrated by construction of a chimaeric protein consisting of the SLH domain of SbsB, the S-layer protein of Geobacillus stearothermophilus PV72/p2 and the C-terminal part of SbpA. In contrast to SbsB or its SLH domain which did not recognize SCWP of B. sphaericus CCM 2177 as binding site, the chimaeric protein showed specific affinity. Deletion of 213 C-terminal amino acids of SbpA had no impact on the square (p4) lattice structure, whereas deletion of 350 amino acids was linked to a change in lattice type from square to oblique (p1).

Amino Acid Sequence↗

PubMed2004

A functional chimaeric S-layer-enhanced green fluorescent protein to follow the uptake of S-layer-coated liposomes into eukaryotic cells.

The chimaeric gene encoding a C-terminally truncated form of the S-layer protein SbpA of Bacillus sphaericus CCM 2177 and the EGFP (enhanced green fluorescent protein) was ligated into plasmid pET28a and cloned and expressed in Escherichia coli. Just 1 h after induction of expression an intense EGFP fluorescence was detected in the cytoplasm of the host cells. Expression at 28 degrees C instead of 37 degrees C resulted in clearly increased fluorescence intensity, indicating that the folding process of the EGFP moiety was temperature sensitive. To maintain the EGFP fluorescence, isolation of the fusion protein from the host cells had to be performed in the presence of reducing agents. SDS/PAGE analysis, immunoblotting and N-terminal sequencing of the isolated and purified fusion protein confirmed the presence of both the S-layer protein and the EGFP moiety. The fusion protein had maintained the ability to self-assemble in suspension and to recrystallize on peptidoglycan-containing sacculi or on positively charged liposomes, as well as to fluoresce. Comparison of fluorescence excitation and emission spectra of recombinant EGFP and rSbpA(31-1068)/EGFP revealed identical maxima at 488 and 507 nm respectively. The uptake of liposomes coated with a fluorescent monomolecular protein lattice of rSbpA(31-1068)/EGFP into HeLa cells was studied by confocal laser-scanning microscopy. The major part of the liposomes was internalized within 2 h of incubation and entered the HeLa cells by endocytosis.

Bacterial Proteins↗

PubMed2003

Crystallization and preliminary structure determination of the C-terminal truncated domain of the S-layer protein SbsC.

The C-terminal truncated form of the S-layer protein SbsC from Geobacillus stearothermophilus, rSbsC(31-844), has been crystallized by the vapour-diffusion method using polyethylene glycol 6000 as a precipitating agent. The crystals diffract to 3 A resolution using synchrotron radiation and belong to space group P2(1), with unit-cell parameters a = 57.24, b = 98.91, c = 108.62 A, beta = 94.34 degrees. One molecule is present in the asymmetric unit, which corresponds to a solvent content of 65%. Native and heavy-atom derivative data have been collected. The Pt derivative yielded two high-occupancy sites per molecule.

Bacterial Proteins↗

PubMed2002

A recombinant bacterial cell surface (S-layer)-major birch pollen allergen-fusion protein (rSbsC/Bet v1) maintains the ability to self-assemble into regularly structured monomolecular lattices and the functionality of the allergen.

The mature crystalline bacterial cell surface (S-layer) protein SbsC of Bacillus stearothermophilus ATCC 12980 comprises amino acids 31-1099 and assembles into an oblique lattice type. As the deletion of up to 179 C-terminal amino acids did not interfere with the self-assembly properties of SbsC, the sequence encoding the major birch pollen allergen (Bet v1) was fused to the sequence encoding the truncated form rSbsC(31-920). The S-layer fusion protein, termed rSbsC/Bet v1, maintained the ability to self-assemble into flat sheets and open-ended cylinders. The presence and the functionality of the fused Bet v1 sequence was proved by blot experiments using BIP1, a monoclonal antibody against Bet v1 and Bet v1-specific IgE-containing serum samples from birch pollen allergic patients. The location and accessibility of the allergen moiety on the outer surface of the S-layer lattice were demonstrated by immunogold labeling of the rSbsC/Bet v1 monolayer, which was obtained by oriented recrystallization of the S-layer fusion protein on native cell wall sacculi. Thereby, the specific interactions between the N-terminal part of SbsC and a distinct type of secondary cell wall polymer were exploited. This is the first S-layer fusion protein described that had retained the specific properties of the S-layer protein moiety in addition to those of the fused functional peptide sequence.

Allergens↗

PubMed2002

Molecular characterization of the S-layer gene, sbpA, of Bacillus sphaericus CCM 2177 and production of a functional S-layer fusion protein with the ability to recrystallize in a defined orientation while presenting the fused allergen.

The nucleotide sequence encoding the crystalline bacterial cell surface (S-layer) protein SbpA of Bacillus sphaericus CCM 2177 was determined by a PCR-based technique using four overlapping fragments. The entire sbpA sequence indicated one open reading frame of 3,804 bp encoding a protein of 1,268 amino acids with a theoretical molecular mass of 132,062 Da and a calculated isoelectric point of 4.69. The N-terminal part of SbpA, which is involved in anchoring the S-layer subunits via a distinct type of secondary cell wall polymer to the rigid cell wall layer, comprises three S-layer-homologous motifs. For screening of amino acid positions located on the outer surface of the square S-layer lattice, the sequence encoding Strep-tag I, showing affinity to streptavidin, was linked to the 5' end of the sequence encoding the recombinant S-layer protein (rSbpA) or a C-terminally truncated form (rSbpA(31-1068)). The deletion of 200 C-terminal amino acids did not interfere with the self-assembly properties of the S-layer protein but significantly increased the accessibility of Strep-tag I. Thus, the sequence encoding the major birch pollen allergen (Bet v1) was fused via a short linker to the sequence encoding the C-terminally truncated form rSpbA(31-1068). Labeling of the square S-layer lattice formed by recrystallization of rSbpA(31-1068)/Bet v1 on peptidoglycan-containing sacculi with a Bet v1-specific monoclonal mouse antibody demonstrated the functionality of the fused protein sequence and its location on the outer surface of the S-layer lattice. The specific interactions between the N-terminal part of SbpA and the secondary cell wall polymer will be exploited for an oriented binding of the S-layer fusion protein on solid supports to generate regularly structured functional protein lattices.

Bacillus↗

PubMed2001

Analysis of the structure-function relationship of the S-layer protein SbsC of Bacillus stearothermophilus ATCC 12980 by producing truncated forms.

The mature surface layer (S-layer) protein SbsC of Bacillus stearothermophilus ATCC 12980 comprises amino acids 31-1099 and self-assembles into an oblique lattice type which functions as an adhesion site for a cell-associated high-molecular-mass exoamylase. To elucidate the structure-function relationship of distinct segments of SbsC, three N- and seven C-terminal truncations were produced in a heterologous expression system, isolated, purified and their properties compared with those of the recombinant mature S-layer protein rSbsC(31-1099). With the various truncated forms it could be demonstrated that the N-terminal part (aa 31-257) is responsible for anchoring the S-layer subunits via a distinct type of secondary cell wall polymer to the rigid cell wall layer, but this positively charged segment is not required for the self-assembly of SbsC, nor for generating the oblique lattice structure. If present, the N-terminal part leads to the formation of in vitro double-layer self-assembly products. Affinity studies further showed that the N-terminal part includes an exoamylase-binding site. Interestingly, the N-terminal part carries two sequences of 6 and 7 aa (AKAALD and KAAYEAA) that were also identified on the amylase-binding protein AbpA of Streptococcus gordonii. In contrast to the self-assembling N-terminal truncation rSbsC(258-1099), two further N-terminal truncations (rSbsC(343-1099), rSbsC(447-1099)) and three C-terminal truncations (rSbsC(31-713), rSbsC(31-844), rSbsC(31-860)) had lost the ability to self-assemble and stayed in the water-soluble state. Studies with the self-assembling C-terminal truncations rSbsC(31-880), rSbsC(31-900) and rSbsC(31-920) revealed that the C-terminal 219 aa can be deleted without interfering with the self-assembly process, while the C-terminal 179 aa are not required for the formation of the oblique lattice structure.

Bacterial Proteins↗

PubMed2000

S-layer gene sbsC of Bacillus stearothermophilus ATCC 12980: molecular characterization and heterologous expression in Escherichia coli.

The cell surface of Bacillus stearothermophilus ATCC 12980 is completely covered with an oblique S-layer lattice. To investigate sequence identities and a common structure-function relationship in S-layer proteins of different B. stearothermophilus wild-type strains, the nucleotide sequence encoding the S-layer protein SbsC of B. stearothermophilus ATCC 12980 was determined by PCR techniques. The entire sbsC sequence showed an ORF of 3297 bp predicted to encode a protein of 1099 aa with a theoretical molecular mass of 115409 Da and an isoelectric point of 5.73. Primer extension analysis suggested the existence of two promoter regions. Amino acid sequence comparison between SbsC and SbsA, a previously characterized S-layer protein of B. stearothermophilus PV72/p6 which assembles into a hexagonally ordered lattice, revealed an identical secretion signal peptide, 85% identity for the N-terminal regions (aa 31-270) which do not carry any S-layer homologous motifs, but only 21% identity for the rest of the sequences. Affinity studies demonstrated that the N-terminal part of SbsC is necessary for recognition of a secondary cell wall polymer. This was in accordance with results obtained in a previous study for SbsA, thus confirming a common functional principle for the N-terminal parts of both S-layer proteins. The sbsC coding region cloned into the pET3a vector without its own upstream region, the signal sequence and the 3' transcriptional terminator led to stable expression in Escherichia coli.

Bacterial Proteins↗

PubMed2000

ISBst12, a novel type of insertion-sequence element causing loss of S-layer-gene expression in Bacillus stearothermophilus ATCC 12980.

The cell surface of the surface layer (S-layer)-carrying strain of Bacillus stearothermophilus ATCC 12980 is completely covered with an oblique lattice composed of the S-layer protein SbsC. In the S-layer-deficient strain, theS-layer gene sbsC was still present but was interrupted by a novel type of insertion sequence (IS) element designated ISBst12. The insertion site was found to be located within the coding region of the sbsC gene, 199 bp downstream from the translation start of SbsC. ISBst12 is 1612 bp long, bounded by 16 bp imperfect inverted repeats and flanked by a directly repeated 8 bp target sequence. ISBst12 contains an ORF of 1446 bp and is predicted to encode a putative transposase of 482 aa with a calculated theoretical molecular mass of 55562 Da and an isoelectric point of 9.13. The putative transposase does not exhibit a typical DDE motif but displays aHis-Arg-Tyr triad characteristic of the active site of integrases from the bacteriophage lambda Int family. Furthermore, two overlapping leucine-zipper motifs were identified at the N-terminal part of the putative transposase. As revealed by Southern blotting, ISBst12 was present in multiple copies in the S-layer-deficient strain as well as in the S-layer-carrying strain. Northern blotting indicated that S-layer gene expression is already inhibited at the transcriptional level, since no sbsC-specific transcript could be identified in the S-layer-deficient strain. By using PCR, ISBst12 was also detected in B. stearothermophilus PV72/p6, in its oxygen-induced strain variant PV72/p2 and in the S-layer-deficient strain PV72/T5.

Amino Acid Sequence↗

PubMedDate not supplied

Generation of a functional monomolecular protein lattice consisting of an s-layer fusion protein comprising the variable domain of a camel heavy chain antibody.

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 rSbpA(31)(-)(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 rSbpA(31)(-)(1068) and cAbs directed against various antigens shall be exploited for building up monomolecular functional protein lattices as required for applications in nanobiotechnology.

Animals↗

PubMedDate not supplied

An S-layer heavy chain camel antibody fusion protein for generation of a nanopatterned sensing layer to detect the prostate-specific antigen by surface plasmon resonance technology.

The bacterial cell surface layer (S-layer) protein of Bacillus sphaericus CCM 2177 assembles into a square lattice structure and recognizes a distinct type of secondary cell wall polymer (SCWP) as the proper anchoring structure in the rigid cell wall layer. For generating a nanopatterned sensing layer with high density and well defined distance of the ligand on the outermost surface, an S-layer fusion protein incorporating the sequence of a variable domain of a heavy chain camel antibody directed against prostate-specific antigen (PSA) was constructed, produced, and recrystallized on gold chips precoated with thiolated SCWP. The S-layer protein moiety consisted of the N-terminal part which specifically recognized the SCWP as binding site and the self-assembly domain. The PSA-specific variable domain of the camel heavy chain antibody was selected by several rounds of panning from a phage display library of an immunized dromedary, and was produced by heterologous expression in Escherichia coli. For construction of the S-layer fusion protein, the 3'-end of the sequence encoding the C-terminally truncated form rSbpA(31)(-)(1068) was fused via a short linker to the 5'-end of the sequence encoding cAb-PSA-N7. The S-layer fusion protein had retained the ability to self-assemble into the square lattice structure. According to the selected fusion site in the SbpA sequence, the cAb-PSA-N7 moiety remained located on the outer surface of the protein lattice. After recrystallization of the S-layer fusion protein on gold chips precoated with thiolated SCWP, the monomolecular protein lattice was exploited as sensing layer in surface plasmon resonance biochips to detect PSA.

Animals↗