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PubMed1990

Detection of Staphylococcus aureus infection by enzyme-linked immunosorbent assay and immunoblotting, using high molecular weight staphylococcal proteins.

Two high molecular weight staphylococcal proteins, fibronectin-binding protein and a Mr 200,000 protein, were investigated as antigens for serodiagnosis of staphylococcal infections. Sera from patients with staphylococcal infections and from controls were subjected to immunoblot analysis with staphylococcal lysate proteins to identify staphylococcal antigens to which patients with staphylococcal infections specifically exhibited antibodies. One such protein was found in the Mr 200,000 region. This protein was purified and used as antigen in ELISA and compared with other antigens, namely fibronectin-binding protein(s) (FNBP, Mr 185,000), alpha-toxin and teichoic acid. Sera from patients with staphylococcal infections contained antibodies to the high molecular weight proteins in higher titers than sera from patients with non-staphylococcal infections or healthy subjects. Based on their amino-acid compositions and different abilities to bind fibronectin it was concluded that the Mr 200,000 protein and FNBP were not identical.

Adolescent↗
PubMed1988

Detection and use of recombinant staphylococcal protein A fusion proteins to localize nucleic-acid-binding domains of proteins.

We developed a rapid method to visualize recombinant staphylococcal protein A fused to segments of another protein and detect DNA-binding proteins simultaneously. Fusion genes were expressed in Escherichia coli to produce fusion proteins of protein A and segments of c-myc oncoprotein. Polypeptides from total E. coli lysates were separated in sodium dodecyl sulfate-polyacrylamide gels and transferred onto nitrocellulose membranes. Without the use of an antigen-specific antibody, protein A/c-myc fusion proteins were visualized by incubation of nitrocellulose membranes with horseradish-peroxidase (HRP)-conjugated immunoglobulin, the Fc region of which directly binds protein A. The sensitivity of detection of the protein A/c-myc fusion proteins was greatly enhanced by incubation of nitrocellulose membranes with rabbit immunoglobulin before incubation with HRP-conjugated goat anti-rabbit antibody. Prior to incubation of nitrocellulose membranes with immunoglobulin, the membranes were incubated with radiolabeled DNA to visualize DNA-binding proteins by autoradiography. Purified staphylococcal protein A does not bind DNA, whereas a segment of c-myc oncoprotein fused to protein A binds DNA in vitro. These methods may be applied to identify nucleic-acid-binding domains of other proteins without prior purification of the fusion proteins.

Binding Sites↗
PubMed1987

Mitogenic activity of staphylococcal protein A is due to contaminating staphylococcal enterotoxins.

Soluble protein A from S. aureus is widely used as a polyclonal activator of human T cells. However, recombinant protein A produced in E. coli does not show any mitogenic properties, although its IgG-binding activity is identical to protein A purified from S. aureus. Antisera against the staphylococcal enterotoxins A and B are able to specifically inhibit the response of human T lymphocytes to protein A from S. aureus. Therefore, the mitogenic principle of this extensively used T cells activator is due to minute contaminations by enterotoxins that are active in picomolar concentrations.

Cells, Cultured↗
PubMed1997

Staphylococcal protein A binding to VH3 encoded immunoglobulins.

Staphylococcal protein A (SPA) is a B-cell superantigen which binds specifically to the variable region of human VH3 encoded antibodies. We undertook to identify the VH3 regions involved in the interaction with SPA by producing mutant antibodies in the baculovirus expression system. We had previously shown that a single amino acid change at position 57 in the CDR2 of a human SPA nonbinding VH3 encoded rheumatoid factor converted it to an SPA binder, implicating CDR2 in SPA binding. When regions of the mutated binder were exchanged with those from a mouse nonbinding antibody, the pattern of SPA binding indicated that residues in FR1, CDR2 and FR3 are involved in the interaction between VH3 encoded antibodies and SPA. In addition, all three regions are simultaneously required for SPA binding to occur. When any one of the three regions was altered, SPA binding was severely disrupted.

Amino Acid Sequence↗
PubMed1998

A study of the interactions between an IgG-binding domain based on the B domain of staphylococcal protein A and rabbit IgG.

The nonantigenic interaction between a recombinant immunoglobulin G (IgG)-binding protein based on the B domain of Protein A from Staphylococcus aureus (termed SpA1) and the Fc fragment of rabbit IgG has been investigated. The contribution to binding of four putative hydrogen bond contacts between SpA1 and IgG-Fc were examined by the individual substitution of the residues in SpA1 involved in these interactions by others unable to form hydrogen bonds. It was found that the most important of the hydrogen bonds involved Tyr 18 which, when replaced by Phe, resulted in a twofold decrease in IgG-binding affinity. The residues of SpA1 proposed to make close, mainly hydrophobic, contacts with Fc were replaced by residues with potential electrostatic charge to establish the importance of the hydrophobic interaction in the complex. The IgG-binding affinities of the mutant proteins were compared to the wild-type protein by a competitive enzyme-linked immunosorbent assay. The replacement of individual hydrophobic residues by His generated a number of novel IgG-binding proteins with reduced binding affinity at pH 5.0 but which maintained strong binding affinities at pH 8.0. The elution profile of human IgG1-Fc (Fc fragment of human IgG1) from a column made from an immobilized two-domain mutant protein shows that the complex dissociates at a higher pH relative to that of the non-mutated protein thus offering favorable elution characteristics.

Animals↗
PubMed1992

Production and secretion of a bifunctional staphylococcal protein A::antiphytochrome single-chain Fv fusion protein in Escherichia coli.

A bifunctional molecule was genetically engineered which contained the secretory signal and four Fc-binding domains of Staphylococcus aureus protein A (FcA), fused to a single-chain Fv (scFv) derived from an immunoglobulin (Ig) G1 mouse monoclonal antibody (AS32) directed against the plant regulatory photoreceptor protein, phytochrome. The FcA::AS32scFv sequence was encoded in a single synthetic gene and expressed as a 60-kDa periplasmic protein in Escherichia coli. The bifunctionality of the fusion protein was established by its ability to bind to both IgG-agarose and phytochrome-sepharose. Growth of cultures, producing the FcA::AS32scFv, at 37 degrees C, resulted in a decrease in the periplasmic accumulation of the fusion protein, and an increased accumulation of an assumed degradation product which retained Fc-binding activity. Growth of cultures at lower temperatures favoured the accumulation of undegraded fusion protein. The recombinant fusion protein could be purified to homogeneity by a simple, rapid chromatography procedure.

Animals↗
PubMed2000

Expression of staphylococcal protein Sbi is induced by human IgG.

Protein Sbi is an IgG- and beta(2) glycoprotein I-binding protein on the surface of Staphylococcus aureus. In most strains, the amount of protein Sbi on the cell surface is very low under normal growth conditions. However, here we show that after growth in the presence of human serum, the amount is significantly increased. In S. aureus strain 8325-4, the observed increase is concentration-dependent and the highest level is found approximately 2 h after serum addition. The active molecule in serum was found to be IgG, which causes an increase of surface-located protein Sbi in S. aureus strain 8325-4, in the clinical isolates tested as well as in protein A-negative mutants. Thus, the results suggest that binding of IgG to protein Sbi upregulates protein Sbi synthesis.

Animals↗
PubMed1988

Staphylococcal protein A (SpA): a potent in vivo chemotactic agent for rat leucocytes.

Staphylococcal protein A had strong chemotactic attraction in vivo to rat leucocytes. Doses as small as 5 micrograms attracted net leucocytes into experimental pellets in 6 h. 50 micrograms Staphylococcal protein A showed maximum chemotactic activity and does greater or less than 50 micrograms attracted less net leucocytes into experimental pellets. The effect of time on the chemoattraction of 50 micrograms Staphylococcal protein A showed that it was an early chemoattractant. Chemotactic activity for this dose, shown by the chemotactic index, reached a peak at 6 h followed by maximum leucocytic infiltration, and almost disappeared completely at 12 h. Leucocytic migration into control pellets rose from 3 h and reached a peak at 12 h (later than the chemotactic peak). Staphylococcal protein A also showed in this study a "later reaction" from 24 to 36 h, resulting in local inflammation of the test site and rise in cellular response.

Animals↗
PubMed1980

Serological evaluation of human brain tumors using a microradioisotopic staphylococcal protein A assay.

A microradioisotopic staphylococcal protein A assay was applied to the serological evaluation of preoperative sera from brain tumor patients. The assay was performed in Terasaki plates, and a stainless steel punch and multiholed die facilitated sample prearation. Using allogeneic astrocytoma target cells, 15/19 of the brain tumor patients' sera were reactive, compared to 1/21 controls 0/7 nonmalignant brain disease patients' sera were positive, and 5/20 sera from patients with epithelial malignancies. The brain tumor sera were not reactive against a rectal cancer cell line.

Brain Neoplasms↗
PubMed1988

Construction of a recombinant expression plasmid encoding a staphylococcal protein A-ricin A fusion protein.

A recombinant plasmid has been constructed containing the coding regions for a functional fragment of staphylococcal protein A and the entire ricin A chain in tandem in the same reading frame. The recombinant gene has been expressed in Escherichia coli cells to produce a protein with both staphylococcal protein A activity and ricin A chain activity. Such a fusion protein could be used for producing immunotoxins.

Cell Line↗
PubMed2000

Expression, purification and applications of staphylococcal protein A fused to cellulose-binding domain.

Because staphylococcal Protein A (ProtA) binds specifically to IgG, it has been used for many immunological manipulations, most notably antibody purification and diagnostics. Immobilization is required for most of these applications. Here we describe a genetic-engineering approach to immobilizing ProtA on cellulose, by fusing it to cellulose-binding domain (CBD) derived from the cellulose-binding Protein A of Clostridium cellulovorans. The bifunctional fusion protein was expressed in Escherichia coli, recovered on a cellulose column and purified by elution at alkaline pH. ProtA-CBD was used to purify IgG from rabbit serum and its ability to bind IgG from different sources was determined. The bifunctional chimaeric protein can bind up to 23.4 mg/ml human IgG at a ratio of 1 mol of ProtA-CBD/2 mol of human IgG, and can purify up to 11.6 mg/ml rabbit IgG from a serum. The ability to bind functionally active CBD-affinity reagents to cellulosic microtitre plates was demonstrated. Our results indicate that a combination of CBD-affinity reagents and cellulosic microtitre plates is an attractive diagnostics matrix for the following reasons: (i) cellulose exhibits very low non-specific binding; and (ii) CBD-fusion proteins bind directly to cellulose at high density. A unique signal-amplification method was developed based on the ability of ProtA-CBD to link stained cellulose particles to primary antibody in a Western blot.

Animals↗
PubMed1992

A modified vector for the controlled high-level overproduction of staphylococcal protein A fusion proteins in the periplasm of Escherichia coli.

A vector encoding the Staphylococcal protein A was modified by cloning the spa gene, including its signal peptide-encoding sequence, downstream of the translation initiation sites of the phage lambda cro gene and under the control of the temperature-inducible phage lambda pR promoter. The expression from this construct was studied using the Escherichia coli phoA gene as a reporter gene after fusion to the spa gene. Determination of alkaline phosphatase activity, 1 h after temperature induction of expression at 42 degrees C, revealed an 800-fold increase over host strain background level. The presence of the alternating selectable markers on the described vector, pHEMa153, which are essential for efficient oligonucleotide-directed construction of mutations by the gapped duplex DNA method, allows the construction of recombinant and mutated forms of Staphylococcal protein A fusion proteins and efficient expression of spa gene fusions without changing the vector system.

Amino Acid Sequence↗
PubMed1978

The binding of murine immunoglobulins to staphylococcal protein A.

The binding of murine IgG1, IgG2a, IgG2b, IgG3 protein molecules to Staphylococcal A protein is presented. Differential elution by sodium thiocyanate gradients is described. Proteins of the IgG2 class (either a or b subclass) require between 1.5 and 2.0 M for complete elution, whereas, IgG1 proteins are fully eluted with only 0.5 M NaSCN. These differential elution patterns can be utilized to distinguish between, or prepare proteins of different Ig classes. It is proposed that this quantitative rather than qualitative distinction between Ig subclass binding to Staph A, indicates the existence of multiple binding sites per molecule, with different markers of such sites being present on the heavy chain of the different Ig classes.

Animals↗
PubMed2003

The YSIRK-G/S motif of staphylococcal protein A and its role in efficiency of signal peptide processing.

Many surface proteins of pathogenic gram-positive bacteria are linked to the cell wall envelope by a mechanism requiring a C-terminal sorting signal with an LPXTG motif. Surface proteins of Streptococcus pneumoniae harbor another motif, YSIRK-G/S, which is positioned within signal peptides. The signal peptides of some, but not all, of the 20 surface proteins of Staphylococcus aureus carry a YSIRK-G/S motif, whereas those of surface proteins of Listeria monocytogenes and Bacillus anthracis do not. To determine whether the YSIRK-G/S motif is required for the secretion or cell wall anchoring of surface proteins, we analyzed variants of staphylococcal protein A, an immunoglobulin binding protein with an LPXTG sorting signal. Deletion of the YSIR sequence or replacement of G or S significantly reduced the rate of signal peptide processing of protein A precursors. In contrast, cell wall anchoring or the functional display of protein A was not affected. The fusion of cell wall sorting signals to reporter proteins bearing N-terminal signal peptides with or without the YSIRK-G/S motif resulted in hybrid proteins that were anchored in a manner similar to that of wild-type protein A. The requirement of the YSIRK-G/S motif for efficient secretion implies the existence of a specialized mode of substrate recognition by the secretion pathway of gram-positive cocci. It seems, however, that this mechanism is not essential for surface protein anchoring to the cell wall envelope.

Amino Acid Motifs↗
PubMed2002

A highly sensitive assay for proteases using staphylococcal protein A fused with enhanced green fluorescent protein.

Enhanced green fluorescent protein (EGFP) was fused with staphylococcal protein A (SpA) and used as a substrate for proteases. An SpA-EGFP assay was done in three steps: (i) digestion of SpA-EGFP by proteases, (ii) addition of rabbit IgG immobilized on Sepharose beads, and (iii) measurement of the fluorescence intensity of supernatant. The assay was sensitive enough to measure picogram levels of trypsin and chymotrypsin, and may be applicable to various other proteases as one of the most sensitive methods.

Biomarkers↗