Affinity purification of human IgG using immobilised, mutated immunoglobulin-binding domains from protein A of Staphylococcus aureus

1998 ◽  
Vol 26 (3) ◽  
pp. S249-S249 ◽  
Author(s):  
Nicola L. Brown ◽  
Stephen P. Bottomley ◽  
Michael G. Gore
Keyword(s):  
Staphylococcus Aureus ◽  
Affinity Purification ◽  
Protein A ◽  
Human Igg ◽  
Binding Domains ◽  
Immunoglobulin Binding

scholarly journals A Protein A based Staphylococcus aureus vaccine with improved safety

2021 ◽  
Author(s):  
Miaomiao Shi ◽  
Xinhai Chen ◽  
Yan Sun ◽  
Hwan Keun Kim ◽  
Olaf Schneewind ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
B Cells ◽  
Immune Escape ◽  
Protective Efficacy ◽  
Protein A ◽  
Staphylococcal Protein A ◽  
Cell Responses ◽  
Binding Domains ◽  
Ige Receptors ◽  
Immunoglobulin Binding

Exposure to Staphylococcus aureus does not lead to immunity as evidenced by the persistent colonization of one third of the human population. S. aureus immune escape is mediated by factors that preempt complement activation, destroy phagocytes, and modify B and T cell responses. One such factor, Staphylococcal protein A (SpA) encompasses five Immunoglobulin binding domains (IgBDs) that associate with the Fcγ domain to block phagocytosis. IgBDs also associate with the Fab domain of VH3-idiotypic IgM which activates B cells with the resulting secretion of antibodies that cannot bind determinants of S. aureus. SpA crosslinking of VH3-idiotypic IgG and IgE receptors of mast cells and basophils promotes histamine release and anaphylaxis. Previous work demonstrated the safety, immunogenicity, and protective efficacy of SpAKKAA, a variant partially defective for VH3-idiotypic Ig cross-linking, in murine models of S. aureus. Compared to mice (10%), humans produce significantly more VH3-idiotypic B cells (50%), prompting a search for safer SpA variants that may be suitably developed as clinical-grade vaccines for efficacy testing in humans. Here, we report the identification of such variants.

scholarly journals Immunoglobulin-binding domains: Protein L from Peptostreptococcus magnus

2003 ◽  
Vol 31 (3) ◽  
pp. 716-718 ◽  
Author(s):  
N.G. Housden ◽  
S. Harrison ◽  
S.E. Roberts ◽  
J.A. Beckingham ◽  
M. Graille ◽  
...  
Keyword(s):  
Binding Sites ◽  
Protein A ◽  
Cell Wall Protein ◽  
Protein G ◽  
Protein L ◽  
Heavy Chains ◽  
Binding Domains ◽  
Peptostreptococcus Magnus ◽  
Immunoglobulin Binding

Protein L is a multidomain cell-wall protein isolated from Peptostreptococcus magnus. It belongs to a group of proteins that contain repeated domains that are able to bind to Igs without stimulating an immune response, the most characterized of this group being Protein A (Staphylococcus aureus) and Protein G (Streptococcus). Both of these proteins bind predominantly to the interface of CH2-CH3 heavy chains, while Protein L binds exclusively to the VL domain of the κ-chain. The function of these proteins in vivo is not clear but it is thought that they enable the bacteria to evade the host's immune system. Two binding sites for κ-chain on a single Ig-binding domain from Protein L have recently been reported and we give evidence that one site has a 25–55-fold higher affinity for κ-chain than the second site.

scholarly journals The Sbi Protein Is a Multifunctional Immune Evasion Factor of Staphylococcus aureus

2011 ◽  
Vol 79 (9) ◽  
pp. 3801-3809 ◽  
Author(s):  
Emma Jane Smith ◽  
Livia Visai ◽  
Steven W. Kerrigan ◽  
Pietro Speziale ◽  
Timothy J. Foster
Keyword(s):  
Staphylococcus Aureus ◽  
Immune Evasion ◽  
Capsular Polysaccharide ◽  
Cell Envelope ◽  
Protein A ◽  
Biologically Active ◽  
Complement Protein ◽  
Content Type ◽  
Binding Domains ◽  
Terminal Domains

ABSTRACTThe second immunoglobulin-binding protein (Sbi) ofStaphylococcus aureushas two N-terminal domains that bind the Fc region of IgG in a fashion similar to that of protein A and two domains that can bind to the complement protein C3 and promote its futile consumption in the fluid phase. It has been proposed that Sbi helps bacteria to avoid innate immune defenses. By comparing a mutant defective in Sbi with mutants defective in protein A, clumping factor A, iron-regulated surface determinant H, and capsular polysaccharide, it was shown that Sbi is indeed an immune evasion factor that promotes bacterial survival in whole human blood and the avoidance of neutrophil-mediated opsonophagocytosis. Sbi is present in the culture supernatant and is also associated with the cell envelope.S. aureusstrains that expressed truncates of Sbi lacking N-terminal domains D1 and D2 (D1D2) or D3 and D4 (D3D4) or a C-terminal truncate that was no longer retained in the cell envelope were analyzed. Both the secreted and envelope-associated forms of Sbi contributed to immune evasion. The IgG-binding domains contributed only when Sbi was attached to the cell, while only the secreted C3-binding domains were biologically active.

scholarly journals Protein A-Specific Monoclonal Antibodies and Prevention of Staphylococcus aureus Disease in Mice

2012 ◽  
Vol 80 (10) ◽  
pp. 3460-3470 ◽  
Author(s):  
Hwan Keun Kim ◽  
Carla Emolo ◽  
Andrea C. DeDent ◽  
Fabiana Falugi ◽  
Dominique M. Missiakas ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Monoclonal Antibodies ◽  
Immune Responses ◽  
B Cell ◽  
Protein A ◽  
Abscess Formation ◽  
Staphylococcal Protein A ◽  
Content Type ◽  
Humoral Immune ◽  
Binding Domains

ABSTRACTStaphylococcus aureusis a leading cause of human soft tissue infections and bacterial sepsis. The emergence of antibiotic-resistant strains (methicillin-resistantS. aureus[MRSA]) has prompted research into staphylococcal vaccines and preventive measures. The envelope ofS. aureusis decorated with staphylococcal protein A (SpA), which captures the Fcγ portion of immunoglobulins to prevent opsonophagocytosis and associates with the Fab portion of VH3-type B cell receptors to trigger B cell superantigen activity. Nontoxigenic protein A (SpAKKAA), when used as an immunogen in mice, stimulates humoral immune responses that neutralize the Fcγ and the VH3+Fab binding activities of SpA and provide protection from staphylococcal abscess formation in mice. Here, we isolated monoclonal antibodies (MAbs) against SpAKKAAthat, by binding to the triple-helical bundle fold of its immunoglobulin binding domains (IgBDs), neutralize the Fcγ and Fab binding activities of SpA. SpAKKAAMAbs promoted opsonophagocytic killing of MRSA in mouse and human blood, provided protection from abscess formation, and stimulated pathogen-specific immune responses in a mouse model of staphylococcal disease. Thus, SpAKKAAMAbs may be useful for the prevention and therapy of staphylococcal disease in humans.

scholarly journals Protein A Is Released into the Staphylococcus aureus Culture Supernatant with an Unprocessed Sorting Signal

2015 ◽  
Vol 83 (4) ◽  
pp. 1598-1609 ◽  
Author(s):  
Dara P. O'Halloran ◽  
Kieran Wynne ◽  
Joan A. Geoghegan
Keyword(s):  
Staphylococcus Aureus ◽  
Protein A ◽  
Covalent Attachment ◽  
Sortase A ◽  
Reporter Protein ◽  
Extracellular Medium ◽  
Content Type ◽  
Sorting Signal ◽  
Immunoglobulin Binding Protein ◽  
Immunoglobulin Binding

The immunoglobulin binding protein A (SpA) ofStaphylococcus aureusis synthesized as a precursor with a C-terminal sorting signal. The sortase A enzyme mediates covalent attachment to peptidoglycan so that SpA is displayed on the surface of the bacterium. Protein A is also found in the extracellular medium, but the processes involved in its release are not fully understood. Here, we show that a portion of SpA is released into the supernatant with an intact sorting signal, indicating that it has not been processed by sortase A. Release of SpA was reduced when the native sorting signal of SpA was replaced with the corresponding region of another sortase-anchored protein (SdrE). Similarly, a reporter protein fused to the sorting signal of SpA was released to a greater extent than the same polypeptide fused to the SdrE sorting signal. Released SpA protected bacteria from killing in human blood, indicating that it contributes to immune evasion.

scholarly journals The first report on the sortase-mediated display of bioactive protein A from Staphylococcus aureus (SpA) on the surface of the vegetative form of Bacillus subtilis

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Samira Ghaedmohammadi ◽  
Gholamreza Ahmadian
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Bacillus Subtilis ◽  
Binding Capacity ◽  
Surface Display ◽  
Protein A ◽  
Production Costs ◽  
Rabbit Serum ◽  
Experimental Conditions ◽  
Binding Domains

AbstractProtein A (SpA) is one of the most important Staphylococcus aureus cell wall proteins. It includes five immunoglobulin (Ig)-binding domains which can bind to immune complexes through the Fc region of immunoglobulins. The binding of SpA to the polymeric supports can be used to prepare affinity chromatography resins, which are useful for immunoprecipitation (IP) of antibodies. Protein A is also used to purify many anti-cancer antibodies. In this study, SpA was displayed on the surface of Bacillus subtilis cells using a sortase-mediated system to display the target protein to the B. subtilis cell wall. A series of plasmids consisting of cassettes for cell wall-directed protein A as well as negative controls were constructed and transformed into B. subtilis WASD (wprA sigD) cells. SDS-PAGE, western blot, flow cytometry, functional IgG purification assay, and a modified ELISA assay were used to confirm the surface display of SpA and evaluate its function. Semi-quantitative ELISA results showed that the binding capacity of lyophilized Bs-SpA is 100 μg IgG from rabbit serum per 1 mg of cells under optimal experimental conditions. Low production costs, optimal performance, and the use of a harmless strain compared to a similar commercial product predict the possible use of SpA immobilization technology in the future.

scholarly journals Regulation of Rot Expression in Staphylococcus aureus

2007 ◽  
Vol 190 (2) ◽  
pp. 546-554 ◽  
Author(s):  
Hsin-Yeh Hsieh ◽  
Ching Wen Tseng ◽  
George C. Stewart
Keyword(s):  
Staphylococcus Aureus ◽  
Affinity Purification ◽  
Protein A ◽  
Protein Sequencing ◽  
Dependent Manner ◽  
Accessory Gene ◽  
Mobility Shift ◽  
Virulence Gene Expression ◽  
Promoter Fragment ◽  
Repressive Effect

ABSTRACT Repressor of toxins (Rot) is known to be a global regulator of virulence gene expression in Staphylococcus aureus. The function of Rot, but not the transcription of rot, is regulated by the staphylococcal accessory gene regulator (Agr) quorum-sensing system. In addition, the alternative sigma factor (σB) has a repressive effect on rot expression during the postexponential phase of growth. The transcriptional profiles of Rot in σB-positive and σB-negative strains in the postexponential and stationary phases of growth were compared. An upregulation of rot expression was observed during the stationary phase of growth, and this upregulation occurred in a σB-dependent manner. The effects of other staphylococcal transcriptional factors were also investigated. Electrophoretic mobility shift assays revealed that proteins present in staphylococcal lysates retarded the mobility of the rot promoter fragment and that the effect was reduced, but not eliminated, with lysates from strains lacking a functional SarS protein. A modest upregulation of rot expression was also observed in sarS-negative strains. Affinity purification of proteins binding to the rot promoter fragment, followed by N-terminal protein sequencing, identified the SarA and SarR proteins. Primer extension analysis of the rot promoter revealed a number of discreet products. However, these RNA species were not associated with identifiable promoter activity and likely represented RNA breakdown products. Loss of Rot function during the postexponential phase of growth likely involves degradation of the rot mRNA but not the inhibition of rot transcription.

Bacteriophage Surface Display of an Immunoglobulin–binding Domain of Staphylococcus aureus Protein A

1994 ◽  
Vol 12 (2) ◽  
pp. 169-172 ◽  
Author(s):  
Baby M. Djojonegoro ◽  
Michael J. Benedik ◽  
Richard C. Willson
Keyword(s):  
Staphylococcus Aureus ◽  
Surface Display ◽  
Protein A ◽  
Binding Domain ◽  
Immunoglobulin Binding
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