Staphylococcus Aureus Protein A Research Articles

The effects of Staphylococcus aureus protein a (SpA) on the expression of inflammatory cytokines in autoimmune patients and their probable immune response modulation mechanisms

The recombinant Staphylococcal protein A (SpA) is widely used in biotechnology to purify polyclonal and monoclonal IgG antibodies. At very low concentrations, the highly-purified form of the protein A can down-regulate the activation of human B-lymphocytes and macrophages which are the key cells in determining autoimmune diseases. In the present study, the efficiency of three different forms of protein A, including native full-length SpA, the recombinant full-length SpA, and a recombinant truncated form of SpA on the reduction of 4 inflammatory cytokines, including IL-8, IL-1β, TNF-α, and IL-6 by peripheral blood mononuclear cell (PBMCs) were studied and compared to an anti-rheumatoid arthritis commercial drug, Enbrel. The recombinant proteins were expressed in E. coli and the native form of SpA was commercially provided. PBMCs were obtained from adult patients with active rheumatoid arthritis (RA) and healthy control donors. Then, the effect of different doses of the three pure forms of SpA in comparison with Enbrel was investigated by analyzing the expression of selected cytokines using ELISA. The results showed that the truncated form of recombinant SpA significantly reduced the expression of cytokines more effectively than the other full-length formulations as well as the commercial drug Enbrel. In silico analysis shows that in the truncated protein, as the radius of gyration increases, the structure of IgG-binding domains become more open and more exposed to IgG. To summarize, our findings indicate that the truncated form of protein A is the most efficient form of SpA as it significantly decreases the secretion of evaluated cytokines from PBMCs in vitro.

Investigation on the molecular mechanism of SPA interference with osteogenic differentiation of bone marrow mesenchymal stem cells

Binding of Staphylococcus aureus protein A (SPA) to osteoblasts induces apoptosis and inhibits bone formation. Bone marrow-derived mesenchymal stem cells (BMSCs) have the ability to differentiate into bone, fat and cartilage. Therefore, it was important to analyze the molecular mechanism of SPA on osteogenic differentiation. We introduced transcript sequence data to screen out differentially expressed genes (DEGs) related to SPA-interfered BMSC. Protein–protein interaction (PPI) network of DEGs was established to screen biomarkers associated with SPA-interfered BMSC. Receiver operating characteristic (ROC) curve was plotted to evaluate the ability of biomarkers to discriminate between two groups of samples. Finally, we performed GSEA and regulatory analysis based on biomarkers. We identified 321 DEGs. Subsequently, 6 biomarkers (Cenpf, Kntc1, Nek2, Asf1b, Troap and Kif14) were identified by hubba algorithm in PPI. ROC analysis showed that six biomarkers could clearly discriminate between normal differentiated and SPA-interfered BMSC. Moreover, we found that these biomarkers were mainly enriched in the pyrimidine metabolism pathway. We also constructed '71 circRNAs-14 miRNAs-5 mRNAs' and '10 lncRNAs-5 miRNAs-2 mRNAs' networks. Kntc1 and Asf1b genes were associated with rno-miR-3571. Nek2 and Asf1b genes were associated with rno-miR-497-5p. Finally, we found significantly lower expression of six biomarkers in the SPA-interfered group compared to the normal group by RT-qPCR. Overall, we obtained 6 biomarkers (Cenpf, Kntc1, Nek2, Asf1b, Troap, and Kif14) related to SPA-interfered BMSC, which provided a theoretical basis to explore the key factors of SPA affecting osteogenic differentiation.

Functional integration of protein A binding ability to antibody fragments for convenient and tag-free purification

ABSTRACTAlthough the development of small therapeutic antibodies is important, the affinity tags used for their purification often result in heterogeneous production and immunogenicity. In this study, we integrated Staphylococcus aureus protein A (SpA) binding ability into antibody fragments for convenient and tag-free purification. SpA affinity chromatography is used as a global standard purification method for conventional antibodies owing to its high binding affinity to the Fc region. SpA also has a binding affinity for some variable heavy domains (VH) classified in the VH3 subfamily. Through mutagenesis based on alignment and structural modeling results using the SpA-VH3 cocrystal structure, we integrated the SpA-binding ability into the anti-CD3 single-chain Fv. Furthermore, we applied this mutagenesis approach to more complicated small bispecific antibodies and successfully purified the antibodies using SpA affinity chromatography. The antibodies retained their biological function after purification. Integration of SpA-binding ability into conventional antibody fragments simplifies the purification and monitoring of the production processes and, thus, is an ideal strategy for accelerating the development of small therapeutic antibodies. Furthermore, because of its immunoactivity, the anti-CD3 variable region with SpA-binding ability is an effective building block for developing engineered cancer therapeutic antibodies without the Fc region.

Expression and Characterization of Intein-Cyclized Trimer of Staphylococcus aureus Protein A Domain Z.

Staphylococcus aureus protein A (SpA) is an IgG Fc-binding virulence factor that is widely used in antibody purification and as a scaffold to develop affinity molecules. A cyclized SpA Z domain could offer exopeptidase resistance, reduced chromatographic ligand leaching after single-site endopeptidase cleavage, and enhanced IgG binding properties by preorganization, potentially reducing conformational entropy loss upon binding. In this work, a Z domain trimer (Z3) was cyclized using protein intein splicing. Interactions of cyclic and linear Z3 with human IgG1 were characterized by differential scanning fluorimetry (DSF), surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC). DSF showed a 5 ℃ increase in IgG1 melting temperature when bound by each Z3 variant. SPR showed the dissociation constants of linear and cyclized Z3 with IgG1 to be 2.9 nM and 3.3 nM, respectively. ITC gave association enthalpies for linear and cyclic Z3 with IgG1 of -33.0 kcal/mol and -32.7 kcal/mol, and -T∆S of association 21.2 kcal/mol and 21.6 kcal/mol, respectively. The compact cyclic Z3 protein contains 2 functional binding sites and exhibits carboxypeptidase Y-resistance. The results suggest cyclization as a potential approach toward more stable SpA-based affinity ligands, and this analysis may advance our understanding of protein engineering for ligand and drug development.

Development of pepper vein banding virus chimeric virus-like particles for potential diagnostic and therapeutic applications.

Monoclonal antibodies have attracted wide attention in therapeutics owing to their high efficacy, low toxicity, and specific targeting. However, antibodies cannot cross the cell membrane barrier. Therefore, their therapeutic potential is limited to surface-exposed antigens or secreted proteins. In the present investigation, we have developed a chimeric virus-like particle (VLP) of pepper vein banding virus (PVBV) and explored the possibility of using it as a delivery vehicle for antibodies against intracellular antigens as well as for future applications in immunodiagnostics. The chimeric PVBV particles were generated by genetically engineering the B domain of Staphylococcus aureus protein A (SpA) at the N-terminus of the PVBV coat protein (CP). The chimeric VLPs purified by sucrose density gradient centrifugation had ~440-fold higher affinity towards IgG antibody when compared to SpA. Interestingly, the unassembled chimeric CP with the B-domain at the N-terminus (BCP) purified by Ni-NTA chromatography was a monomer, and it had ~45-fold higher affinity towards antibodies compared to SpA. Additionally, the chimeric particles were able to bind and deliver antibodies against both intracellular (α-tubulin) and surface-exposed antigens (CD 20). However, the BCP monomer failed to enter mammalian cells. Thus, for the first time, we have demonstrated that the assembled VLPs are essential for internalization. These results demonstrate the potential of the use of chimeric PVBV VLPs in diagnostics and, more importantly, as nanocarriers for intracellular delivery of antibodies.

Binding of Staphylococcus aureus Protein A to von Willebrand Factor Is Regulated by Mechanical Force.

Binding of Staphylococcus aureus to the large plasma glycoprotein von Willebrand factor (vWF) is controlled by hydrodynamic flow conditions. Currently, we know little about the molecular details of this shear-stress-dependent interaction. Using single-molecule atomic force microscopy, we demonstrate that vWF binds to the S. aureus surface protein A (SpA) via a previously undescribed force-sensitive mechanism. We identify an extremely strong SpA-vWF interaction, capable of withstanding forces of ∼2 nN, both in laboratory and in clinically relevant methicillin-resistant S. aureus (MRSA) strains. Strong bonds are activated by mechanical stress, consistent with flow experiments revealing that bacteria adhere in larger amounts to vWF surfaces when the shear rate is increased. We suggest that force-enhanced adhesion may involve conformational changes in vWF. Under force, elongation of vWF may lead to the exposure of a high-affinity cryptic SpA-binding site to which bacteria firmly attach. In addition, force-induced structural changes in the SpA domains may also promote strong, high-affinity binding. This force-regulated interaction might be of medical importance as it may play a role in bacterial adherence to platelets and to damaged blood vessels.IMPORTANCEStaphylococcus aureus protein A (SpA) binds to von Willebrand factor (vWF) under flow. While vWF binding to SpA plays a role in S. aureus adherence to platelets and endothelial cells under shear stress, the molecular basis of this stress-dependent interaction has not yet been elucidated. Here we show that the SpA-vWF interaction is regulated by a new force-dependent mechanism. The results suggest that mechanical extension of vWF may lead to the exposure of a high-affinity cryptic SpA-binding site, consistent with the shear force-controlled functions of vWF. Moreover, strong binding may be promoted by force-induced structural changes in the SpA domains. This study highlights the role of mechanoregulation in controlling the adhesion of S. aureus and shows promise for the design of small inhibitors capable of blocking colonization under high shear stress.

An engineered Staphylococcal Protein A based ligand: Production, characterization and potential application for the capture of Immunoglobulin and Fc-fusion proteins

In antibody purification processes, affinity chromatography has been used with Staphylococcus aureus protein A (SpA) as the main ligand. In this work, we present a novel Staphylococcal Protein A (AviPure thereafter), a synthetic ligand analogue based on native SpA B domain, with a molecular weight of approximately 14 kDa.The binding affinity of mAbs to AviPure was evaluated using Surface Plasmon Resonance (SPR) and affinity chromatography methods. The equilibrium dissociation constant (KD) between the AviPure and mAbs was systematically measured using 1:1 (Langmuir) model and found to be 4.7 × 10−8 M, with constant of dissociation at kd ≤ 1.0 × 10−3 s−1 and ka being 3.1 × 104 M−1 s−1. When immobilized on Sepharose, the AviPure ligand density was 429 nmol/g moist weight resin and was able to effectively bind immunoglobulin and Fc fragment samples with higher affinity and the most effective flow rate when using ligand - Sepharose beads was at 75 cm/h giving the dynamic binding capacity of 53 mg/mL and 91% recovery of IgG.Suitable ligands used in affinity purification should have a KD ≤ 10−6 M and a dissociation rate (ka) averaging 10−3 M−1 s−1 with the kd ranging between 103 – 108 M−1. Therefore, the AviPure ligand can be used as an alternative to the standard protein A ligand in the purification of mAbs and Fc-fused proteins.

Wireless poly(dimethylsiloxane) quartz-crystal-microbalance biosensor chip fabricated by nanoimprint lithography for micropump integration aiming at application in lab-on-a-chip

We developed a quartz-crystal-microbalance (QCM) biosensor chip that operates wirelessly via electromagnetic waves, using poly(dimethylsiloxane) (PDMS). An AT-cut quartz oscillator (22–30 µm) is packaged in a microchannel, where it is supported by micropillars without mechanical fixing. As a result, the quartz oscillator is little affected by the thermal stress caused by the difference in the thermal expansion coefficients of the components, and the leakage of the vibration energy of the quartz oscillator is reduced. Consequently, high-frequency (∼56 MHz) measurement with a stable baseline (±∼2 ppm) is realized. We succeeded in repeatedly monitoring the binding reaction between immunoglobulin G (IgG) and Staphylococcus aureus protein A (SPA) with the quartz oscillator on which SPA molecules were immobilized nonspecifically. In addition, the affinity between SPA and IgG was calculated from the association and dissociation curves, and the usefulness of our wireless PDMS QCM biosensor was demonstrated.

Intranasal inoculate of influenza virus vaccine against lethal virus challenge

Vaccine adjuvants are essential for enhancing immune responses during vaccination. However, only a limited number of safe and effective adjuvants, especially mucosal adjuvants, are available for use in vaccines. The development of a practically applicable mucosal adjuvant is therefore urgently needed. Here, we showed that the non-toxic CTA1-DD adjuvant, which combined the full enzymatic activity of the A1 subunit of cholera toxin (CT) with two immunoglobulin-binding domains of Staphylococcus aureus protein A (SpA), promoted mucosal and systemic humoral and cell-mediated immune responses following intranasal administration with H1N1 split vaccine in mice. We demonstrated that CTA1-DD-adjuvant vaccine provided 100% protection against mortality and greatly reduced morbidity in a mouse model. We also showed that addition of CTA1-DD to the vaccine elicited significantly higher hemagglutination inhibition titers and IgG antibodies in sera than alum adjuvant. Furthermore, CTA1-DD significantly promoted the production of mucosal secretory IgA in lung lavages and vaginal lavages. We also showed that CTA1-DD could be used as a mucosal adjuvant to enhance T cell responses. Our results clearly indicated that CTA1-DD contributed to the elicitation of a protective cell-mediated immune response required for efficacious vaccination against influenza virus, which suggested that this adjuvant could be explored further as a clinically safe mucosal vaccine adjuvant for respiratory diseases and other mucosal diseases.

Design of bio-inspired silica-encapsulated protein A for improved immunoprecipitation assays

Staphylococcus aureus Protein A (SpA) has a high affinity to the Fc region of antibodies (Abs), and SpA-immobilized matrices are widely used for Ab purification or immunoprecipitation (IP) assays. Here, we employed a bio-inspired silica-encapsulation method to improve the Ab-binding ability of an SpA-immobilized matrix. Two silica-forming peptides (SFPs), namely R5 and EctP1, were separately introduced at the C-terminus of SpA to generate two recombinant fusion proteins (SpA-SFPs) with auto-silicifying abilities. When SpA-SFPs were employed as Ab-binders on a solid surface (96-well plate), they showed an effective Ab-binding ability and a better performance than intact SpA. A high binding ability was observed even when an SFP-mediated SpA silica matrix (SpA-SFP@SiO2) was prepared. SpA-SFP@SiO2 showed a higher performance than commercially obtained SpA-Agarose particles and no loss of matrices. Moreover, in IP assays, SpA-SFP@SiO2 showed an approximately 300% higher precipitation of target protein than the commercial SpA-Agarose product when a small amount of cell lysate was used. These findings demonstrated that SpA-SFP could be useful for the development of an efficient immunoassay system.

Immuno-biosensor for Detection of CD20-Positive Cells Using Surface Plasmon Resonance.

Purpose: Surface plasmon resonance (SPR) sensing confers a real-time assessment of molecular interactions between biomolecules and their ligands. This approach is highly sensitive and reproducible and could be employed to confirm the successful binding of drugs to cell surface targets. The specific affinity of monoclonal antibodies (MAb) for their target antigens is being utilized for development of immuno-sensors and therapeutic agents. CD20 is a surface protein of B lymphocytes which has been widely employed for immuno-targeting of B-cell related disorders. In the present study, binding ability of an anti-CD20 MAb to surface antigens of intact target cells was investigated by SPR technique.Methods: Two distinct strategies were used for immobilization of the anti-CD20 MAb onto gold (Au) chips. MUA (11-mercaptoundecanoic acid) and Staphylococcus aureus protein A (SpA) were the two systems used for this purpose. A suspension of CD20-positive Raji cells was injected in the analyte phase and the resulting interactions were analyzed and compared to those of MOLT-4 cell line as CD20-negative control.Results: Efficient binding of anti-CD20 MAb to the surface antigens of Raji cell line was confirmed by both immobilizing methods, whereas this MAb had not a noticeable affinity to the MOLT-4 cells.Conclusion: According to the outcomes, the investigated MAb had acceptable affinity and specificity to the target antigens on the cell surface and could be utilized for immuno-detection of CD20-positive intact cells by SPR method.

Staphylococcusaureus Protein A induces osteoclastogenesis via the NF‑κB signaling pathway.

Staphylococcus aureus (S. aureus) is the most common organism causing osteomyelitis, and Staphylococcus aureus protein A (SpA) is an important virulence factor anchored in its cell wall. However, the precise mechanisms underlying the bone loss caused by SpA have not been well understood. The present study aimed to investigate the effect of SpA on osteoclast differentiation, and the probable mechanism was investigated. Raw264.7 cells were treated with SpA in the absence or presence of receptor-activated (NF)-κB ligand for 5 days, and morphological and biochemical assays were used to assess osteoclastogenesis and explore the underlying mechanisms. Data demonstrated that SpA induced osteoclast differentiation and promoted bone resorption in a dose-dependent manner in the absence or presence of RANKL. In addition, the expression of osteoclast-specific genes, such as the tartrate resistant acid phosphatase, matrix metalloproteinase-9, cathepsin K, calcitonin receptors and d2 isoform of the vacuolar ATPase Vo domain, were enhanced by SpA. Furthermore, the SpA-induced osteoclast differentiation was associated with the degradation of inhibitor of κB-α, phosphorylation of NF-κB p65 and increased expression of nuclear factor of activated T-cells. However, by treatment with JSH-23, an NF-κB inhibitor, the formation of osteoclast-like cells and resorption pits was significantly reduced, and the expression of osteoclast-specific genes was also inhibited. Collectively, in the present study SpA induced osteoclast differentiation, promoted bone resorption, and the NF-κB signaling pathway was involved in this process.

In situ monitoring and optimization of CuAAC-mediated protein functionalization of biosurfaces

With the current trend to miniaturize bioactive surfaces to micro- or nanometer scale, the strategy of immobilization becomes more important. Therefore, there is a growing need for protein immobilization techniques that create both stable and homogeneously covered surfaces in a reproducible way. One of the most promising methods to achieve this is the combination of biological receptors with ‘click’ chemistry, like the Copper catalyzed Alkyne Azide Cycloaddition (CuAAC). This work presents a full optimization of all aspects of the ‘click’ chemistry reaction between proteins and surfaces in order to create covalently and homogeneously covered biosurfaces. The coupling procedure is monitored by in situ ellipsometry, a unique characterization technique that offers the opportunity to quantify minute amounts of surface coupled protein mass in real-time. The optimization involves the azidification of a solid silicon support, the alkynation of two proteins, Staphylococcus aureus Protein A (SpA) and Maltose Binding Protein (MBP), as well as the development of a highly reproducible CuAAC ‘click’ coupling protocol. Using the here optimized protocol, active and reproducible biolayers can be created rapidly. The proposed surface biofunctionalization method combined with ellipsometry forms a unique and promising platform towards the development of highly sensitive, accurate biosensors.

Protein a Immunoadsorption May Hamper the Decision to Transplant Due to Interference With CDC Crossmatch Results.

Transplanting immunized patients requires immunological monitoring in the pretransplant phase to follow reduction of donor specific HLA antibodies (DSA) after Staphylococcus aureus protein A (SPA) immunoadsorption (IA) or therapeutic plasma exchange followed by IVIG and Rituximab administration. Pretreatment aims to significantly reduce DSA strength. The Tissue Typing Lab at Aarhus University Hospital performs immunological monitoring of approximately 150 kidney transplantation patients per year from two transplant centers. From 2012 to 2013, we experienced seven patients desensitized using SPA IA, initially presenting negative cytotoxic complement dependent (CDC) T-cell crossmatches but positive B and T cell flowcytometric crossmatch, who despite significant DSA reduction developed weakly positive CDC T-cell crossmatch shortly prior to transplantation. We hypothesised that leached SPA during IA could be the cause, as the complication was not observed in patients who received plasma exchanges. We found that the positive CDC was not donor specific and SPA column material incubated with control serum reproduced a positive CDC T-cell crossmatch. Finally, we detected leached SPA in one of the patient samples using a highly sensitive time-resolved fluorescent assay. In conclusion, the results emphasize the importance of carefully considering CDC crossmatch results subsequent to IA, before a planned transplantation is either postponed or cancelled. J. Clin. Apheresis 32:163-169, 2017. © 2016 Wiley Periodicals, Inc.

Intracellular delivery of antibodies by chimeric Sesbania mosaic virus (SeMV) virus like particles.

The therapeutic potential of antibodies has not been fully exploited as they fail to cross cell membrane. In this article, we have tested the possibility of using plant virus based nanoparticles for intracellular delivery of antibodies. For this purpose, Sesbania mosaic virus coat protein (CP) was genetically engineered with the B domain of Staphylococcus aureus protein A (SpA) at the βH-βI loop, to generate SeMV loop B (SLB), which self-assembled to virus like particles (VLPs) with 43 times higher affinity towards antibodies. CP and SLB could internalize into various types of mammalian cells and SLB could efficiently deliver three different monoclonal antibodies–D6F10 (targeting abrin), anti-α-tubulin (targeting intracellular tubulin) and Herclon (against HER2 receptor) inside the cells. Such a mode of delivery was much more effective than antibodies alone treatment. These results highlight the potential of SLB as a universal nanocarrier for intracellular delivery of antibodies.

Bacteria-mimicking nanoparticle surface functionalization with targeting motifs.

In recent years, surface modification of nanocarriers with targeting motifs has been explored to modulate delivery of various diagnostic, sensing and therapeutic molecular cargo to desired sites of interest in in vitro bioengineering platforms and in vivo pathologic tissue. However, most surface functionalization approaches are often plagued by complex chemical modifications and effortful purifications. To resolve such challenges, this study demonstrates a unique method to immobilize antibodies that can act as targeting motifs on the surfaces of nanocarriers, inspired by a process that bacteria use for immobilization of the host's antibodies. We hypothesized that alkylated Staphylococcus aureus protein A (SpA) would self-assemble with micelles and subsequently induce stable coupling of antibodies to the micelles. We examined this hypothesis by using poly(2-hydroxyethyl-co-octadecyl aspartamide) (PHEA-g-C18) as a model polymer to form micelles. The self-assembly between the micelles and alkylated SpA became more thermodynamically favorable by increasing the degree of substitution of octadecyl chains to PHEA-g-C18, due to a positive entropy change. Lastly, the mixing of SpA-PA-coupled micelles with antibodies resulted in the coating of micelles with antibodies, as confirmed with a fluorescence resonance energy transfer (FRET) assay. The micelles coated with antibodies to VCAM-1 or integrin αv displayed a higher binding affinity to substrates coated with VCAM-1 and integrin αvβ3, respectively, than other controls, as evaluated with surface plasmon resonance (SPR) spectroscopy and a circulation-simulating flow chamber. We envisage that this bacteria-inspired protein immobilization approach will be useful to improve the quality of targeted delivery of nanoparticles, and can be extended to modify the surface of a wide array of nanocarriers.

In vitro molecular evolution yields an NEIBM with a potential novel IgG binding property.

Staphylococcus aureus protein A (SpA) and protein G of groups C and G streptococci (SpG) are two well-defined bacterial immunoglobulin (Ig)-binding proteins (IBPs) with high affinity for specific sites on IgG from mammalian hosts. Both SpA and SpG contain several highly-homologous IgG-binding domains, each of which possesses similar binding characteristic of the whole corresponding proteins. Whether specific combinations of these domains could generate a molecule with novel IgG-binding properties remained unknown. We constructed a combinatorial phage library displaying randomly-rearranged A, B, C, D and E domains of SpA as well as the B2 (G2) and B3 (G3) domains of SpG. In vitro molecular evolution directed by human, rabbit, bovine, or goat polyclonal IgGs and four subclasses of mouse monoclonal IgGs generated one common combination, D-C-G3. A series of assays demonstrated that D-C-G3 exhibited a potential novel IgG binding property that was obviously different from those of both parent proteins. This study provides an example of successful protein engineering through in vitro molecular evolution and useful approaches for structure and function studies of IBPs.

Highly stable aptamers selected from a 2′-fully modified fGmH RNA library for targeting biomaterials

When developed as targeting ligands for the in vivo delivery of biomaterials to biological systems, RNA aptamers immediately face numerous obstacles, in particular nuclease degradation and post-selection 2′ modification. This study aims to develop a novel class of highly stable, 2′-fully modified RNA aptamers that are ideal for the targeted delivery of biomaterials. We demonstrated the facile transcription of a fGmH (2′-F-dG, 2′-OMe-dA/dC/dU) RNA library with unexpected hydrophobicity, the direct selection of aptamers from a fGmH RNA library that bind Staphylococcus aureus Protein A (SpA) as a model target, and the superior nuclease and serum stability of these aptamers compared to 2′-partially modified RNA variants. Characterizations of fGmH RNA aptamers binding to purified SpA and to endogenous SpA present on the surface of S. aureus cells demonstrate fGmH RNA aptamer selectivity and stability. Significantly, fGmH RNA aptamers were able to functionalize, stabilize, and specifically deliver aggregation-prone silver nanoparticles (AgNPs) to S. aureus with SpA-dependent antimicrobial effects. This study describes a novel aptamer class with considerable potential to improve the in vivo applicability of nucleic acid-based affinity molecules to biomaterials.

Molecular Basis for the Dissociation Dynamics of Protein A-Immunoglobulin G1 Complex

Staphylococcus aureus protein A (SpA) is the most popular affinity ligand for immunoglobulin G1 (IgG1). However, the molecular basis for the dissociation dynamics of SpA-IgG1 complex is unclear. Herein, coarse-grained (CG) molecular dynamics (MD) simulations with the Martini force field were used to study the dissociation dynamics of the complex. The CG-MD simulations were first verified by the agreement in the structural and interactional properties of SpA and human IgG1 (hIgG1) in the association process between the CG-MD and all-atom MD at different NaCl concentrations. Then, the CG-MD simulation studies focused on the molecular insight into the dissociation dynamics of SpA-hIgG1 complex at pH 3.0. It is found that there are four steps in the dissociation process of the complex. First, there is a slight conformational adjustment of helix II in SpA. This is followed by the phenomena that the electrostatic interactions provided by the three hot spots (Glu143, Arg146 and Lys154) of helix II of SpA break up, leading to the dissociation of helix II from the binding site of hIgG1. Subsequently, breakup of the hydrophobic interactions between helix I (Phe132, Tyr133 and His137) in SpA and hIgG1 occurs, resulting in the disengagement of helix I from its binding site of hIgG1. Finally, the non-specific interactions between SpA and hIgG1 decrease slowly till disappearance, leading to the complete dissociation of the SpA-hIgG1 complex. This work has revealed that CG-MD coupled with the Martini force field is an effective method for studying the dissociation dynamics of protein-protein complex.

Recognition and enrichment specificity of Fe3O4 magnetic nanoparticles surface modified by chitosan and Staphylococcus aureus enterotoxins A antiserum

Fe3O4 modified by chitosan (CTS) and Staphylococcus aureus enterotoxin A (SEA) antiserum immunomagnetic beads (Fe3O4-CTS-SEA-IB) that targeted enrich SEA were prepared in aqueous solution and using CTS and SEA antiserum as surface modification stabilizer. Recognition experiments of the prepared Fe3O4-CTS-SEA-IB on SEA were conducted to determine adsorption capacity and recognition specificity. The results showed that the SEA antiserum were prepared and successfully conjugated onto Fe3O4-modificated CTS magnetic beads. The Fe3O4-CTS-SEA immunomagnetic beads displayed a high adsorption capacity and recognition specificity for SEA, and the adsorption quantity could reach 6.48×10−3μmol/g. The specificity evaluation results showed that the Fe3O4-CTS-SEA immunomagnetic beads had high enrichment and affinity property for SEA compared to SEB (Staphylococcus aureus enterotoxin B) and SPA (Staphylococcus aureus protein A).