In Vitro Removal of Human Igg by Pseudobiospecific Affinity Membrane Filtration on a Large Scale. A Preliminary Report

1995 ◽  
Vol 18 (7) ◽  
pp. 392-398 ◽  
Author(s):  
S.M.A. Bueno ◽  
K. Haupt ◽  
M.A. Vijayalakshmi
Keyword(s):  
Membrane Filtration ◽  
Large Scale ◽  
Binding Capacity ◽  
Protein A ◽  
High Capacity ◽  
Ether Linkage ◽  
Human Igg ◽  
Affinity Membrane ◽  
Affinity Membranes

We have developed a pseudobiospecific affinity membrane device for selective removal of human IgG from plasma or serum in vitro for clinical apheresis application. The pseudobiospecific affinity ligand L-histidine was immobilized through an ether linkage onto poly(ethylenevinyl alcohol) hollow fiber cartridge. The obtained affinity membranes showed high selectivity for IgG adsorption from untreated human serum. These membranes are able to adsorb lgG1, lgG2, lgG3 if Mops buffer is used, and more selectively lgG1 and lgG3 in Tris-HCl buffer. With respect to the binding capacity, the pseudobiospecific affinity membrane used showed a higher capacity as compared to protein A-membranes described in the literature. Due to the high capacity, specificity and stability of the histidine affinity membranes, in addition to their lower cost, the approach proposed in this paper may offer a useful alternative to protein A based devices in the treatment of immune-related diseases.

scholarly journals Optimization of Sequence, Display, and Mode of Operation of IgG-Binding Peptide Ligands to Develop Robust, High-Capacity Affinity Adsorbents That Afford High IgG Product Quality

2019 ◽  
Vol 20 (1) ◽  
pp. 161 ◽  
Author(s):  
Tuhidul Islam ◽  
Amith D. Naik ◽  
Yasuhiro Hashimoto ◽  
Stefano Menegatti ◽  
Ruben G. Carbonell
Keyword(s):  
Binding Capacity ◽  
Protein A ◽  
Chinese Hamster ◽  
High Capacity ◽  
Alkaline Solutions ◽  
Peptide Ligand ◽  
Dynamic Binding ◽  
Ligand Density ◽  
Host Cell Proteins ◽  
Igg Binding

This work presents the use of peptide ligand HWRGWV and its cognate sequences to develop affinity adsorbents that compete with Protein A in terms of binding capacity and quality of the eluted product. First, the peptide ligand was conjugated to crosslinked agarose resins (WorkBeads) at different densities and using different spacer arms. The optimization of ligand density and display resulted in values of static and dynamic binding capacity of 85 mg/mL and 65 mg/mL, respectively. A selected peptide-WorkBeads adsorbent was utilized for purifying Mabs from Chinese Hamster Ovary (CHO) cell culture supernatants. The peptide-WorkBeads adsorbent was found able to withstand sanitization with strong alkaline solutions (0.5 M NaOH). The purity of the eluted product was consistently higher than 95%, with logarithmic removal value (LRV) of 1.5 for host cell proteins (HCPs) and 4.0 for DNA. HCP clearance was significantly improved by adding a post-load washing step with either 0.1 M Tris HCl pH 9 or 1 M NaCl. The cognate peptide of HWRGWV, constructed by replacing arginine (R) with citrulline, further increased the HCP LRV to 2.15. The peptide-based adsorbent also showed a remarkable performance in terms of removal of Mab aggregates; unlike Protein A, in fact, HWRGWV was found to bind only monomeric IgG. Collectively, these results demonstrate the potential of peptide-based adsorbents as alternative to Protein A for the purification of therapeutic antibodies.

scholarly journals Staphylococcus aureus Protein A Promotes Immune Suppression

mBio ◽  
2013 ◽  
Vol 4 (5) ◽  
Author(s):  
Scott D. Kobayashi ◽  
Frank R. DeLeo
Keyword(s):  
Staphylococcus Aureus ◽  
B Cell ◽  
Immune Evasion ◽  
Bacterial Infections ◽  
Binding Capacity ◽  
Protein A ◽  
Strong Support ◽  
The United States ◽  
Content Type

ABSTRACTStaphylococcus aureusis a prominent cause of human infections worldwide and is notorious for its ability to acquire resistance to antibiotics. Methicillin-resistantS. aureus(MRSA), in particular, is endemic in hospitals and is the most frequent cause of community-associated bacterial infections in the United States. Inasmuch as treatment options for severe MRSA infections are limited, there is need for a vaccine that protects against such infections. However, recent efforts to generate a staphylococcal vaccine have met with little success in human clinical trials. These failures are somewhat puzzling, since the vaccine antigens tested promote opsonophagocytosisin vitroand confer protection in animal infection models. One possibility is that the pathogen inhibits (and/or fails to elicit) the development of protective immunity in humans. Indeed,S. aureusproduces numerous molecules that can potentially promote immune evasion, including protein A (SpA), an immunoglobulin (Ig)-binding protein present on the bacterial surface and freely secreted into the extracellular environment. SpA binds the Fc region of antibody and the Fab regions of the B-cell receptor, processes that are known to block opsonophagocytosis and cause B-cell deathin vitro. In a recent study, Falugi et al. [F. Falugi, H. K. Kim, D. M. Missiakas, and O. Schneewind, mBio 4(5):e00575-13, 2013] showed that vaccination withspamutantS. aureusstrains lacking antibody Fc- and/or Fab-binding capacity protects against subsequent challenge with the USA300 epidemic strain. The findings provide strong support for the idea that SpA promotesS. aureusimmune evasionin vivoand form the foundation for a new approach in our efforts to develop a vaccine that prevents severeS. aureusinfections.

Polyethyleneimine-facilitated high-capacity boronate affinity membrane and its application for the adsorption and enrichment of cis-diol-containing molecules

RSC Advances ◽  
2016 ◽  
Vol 6 (49) ◽  
pp. 43648-43655 ◽  
Author(s):  
Juan Ma ◽  
Chaozhan Wang ◽  
Yinmao Wei
Keyword(s):  
High Capacity ◽  
Adsorption And Desorption ◽  
Affinity Membrane ◽  
Affinity Membranes ◽  
First Time

High capacity boronate affinity membranes were prepared for the first time, the membranes possess good selectivity, faster adsorption and desorption speed towards cis-diol-containing molecules.

scholarly journals Impacts of sarA and agr in Staphylococcus aureus Strain Newman on Fibronectin-Binding Protein A Gene Expression and Fibronectin Adherence Capacity In Vitro and in Experimental Infective Endocarditis

2004 ◽  
Vol 72 (3) ◽  
pp. 1832-1836 ◽  
Author(s):  
Yan-Qiong Xiong ◽  
Arnold S. Bayer ◽  
Michael R. Yeaman ◽  
Willem van Wamel ◽  
Adhar C. Manna ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Binding Capacity ◽  
Protein A ◽  
Aureus Strain ◽  
Global Regulators ◽  
Fibronectin Binding Protein ◽  
Regulatory Loci ◽  
Fibronectin Binding

ABSTRACT We investigated the impacts of sarA and agr on fnbA expression and fibronectin-binding capacity in Staphylococcus aureus in vitro and in experimental endocarditis. Although sarA up-regulated and agr down-regulated both fnbA expression and fibronectin binding in vitro and in vivo, fnbA expression was positively regulated in the absence of both global regulators. Thus, additional regulatory loci contribute to fnbA regulation and fibronectin-binding capacities in S. aureus.

scholarly journals Focused peptide library screening as a route to a superior affinity ligand for antibody purification

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Krištof Bozovičar ◽  
Barbara Jenko Bizjan ◽  
Anže Meden ◽  
Jernej Kovač ◽  
Tomaž Bratkovič
Keyword(s):  
Binding Capacity ◽  
Protein A ◽  
Downstream Processing ◽  
Phage Display Library ◽  
Enrichment Ratio ◽  
Peptide Ligand ◽  
Staphylococcal Protein A ◽  
Dynamic Binding ◽  
Linear Peptide

AbstractAffinity chromatography is the linchpin of antibody downstream processing and typically relies on bacterial immunoglobulin (Ig)-binding proteins, epitomized by staphylococcal protein A-based ligands. However, such affinity ligands are fairly costly and suffer from chemical instability, leading to ligand denaturation and leaching from chromatographic support. Innovations in this area are aimed at developing robust and highly selective antibody ligands capable of withstanding harsh column sanitization conditions. We report the development and first-stage characterization of a selective short linear peptide ligand of the IgG Fc region capable of capturing all four IgG subclasses. The ligand was discovered through in vitro directed evolution. A focused phage-display library based on a previously identified peptide lead was subjected to a single-round screen against a pool of human IgG. The hits were identified with next-generation sequencing and ranked according to the enrichment ratio relative to their frequency in the pre-screened library. The top enriched peptide GSYWYNVWF displaying highest affinity for IgG was coupled to bromohydrin-activated agarose beads via a branched linker. The resulting affinity matrix was characterized with a dynamic binding capacity of approx. 43 mg/mL, on par with commercially employed protein A-based resin.

scholarly journals Redox Coenzyme F420 Biosynthesis in Thermomicrobia Involves Reduction by Stand-Alone Nitroreductase Superfamily Enzymes

2020 ◽  
Vol 86 (12) ◽  
Author(s):  
Daniel Braga ◽  
Mahmudul Hasan ◽  
Tabea Kröber ◽  
Daniel Last ◽  
Gerald Lackner
Keyword(s):  
Large Scale ◽  
Reductase Activity ◽  
Protein A ◽  
Model Organisms ◽  
Gram Negative Bacteria ◽  
Scale Production ◽  
Metabolome Analysis ◽  
Gram Negative ◽  
Content Type

ABSTRACT Coenzyme F420 is a redox cofactor involved in hydride transfer reactions in archaea and bacteria. Since F420-dependent enzymes are attracting increasing interest as tools in biocatalysis, F420 biosynthesis is being revisited. While it was commonly accepted for a long time that the 2-phospho-l-lactate (2-PL) moiety of F420 is formed from free 2-PL, it was recently shown that phosphoenolpyruvate is incorporated in Actinobacteria and that the C-terminal domain of the FbiB protein, a member of the nitroreductase (NTR) superfamily, converts dehydro-F420 into saturated F420. Outside the Actinobacteria, however, the situation is still unclear because FbiB is missing in these organisms and enzymes of the NTR family are highly diversified. Here, we show by heterologous expression and in vitro assays that stand-alone NTR enzymes from Thermomicrobia exhibit dehydro-F420 reductase activity. Metabolome analysis and proteomics studies confirmed the proposed biosynthetic pathway in Thermomicrobium roseum. These results clarify the biosynthetic route of coenzyme F420 in a class of Gram-negative bacteria, redefine functional subgroups of the NTR superfamily, and offer an alternative for large-scale production of F420 in Escherichia coli in the future. IMPORTANCE Coenzyme F420 is a redox cofactor of Archaea and Actinobacteria, as well as some Gram-negative bacteria. Its involvement in processes such as the biosynthesis of antibiotics, the degradation of xenobiotics, and asymmetric enzymatic reductions renders F420 of great relevance for biotechnology. Recently, a new biosynthetic step during the formation of F420 in Actinobacteria was discovered, involving an enzyme domain belonging to the versatile nitroreductase (NTR) superfamily, while this process remained blurred in Gram-negative bacteria. Here, we show that a similar biosynthetic route exists in Thermomicrobia, although key biosynthetic enzymes show different domain architectures and are only distantly related. Our results shed light on the biosynthesis of F420 in Gram-negative bacteria and refine the knowledge about sequence-function relationships within the NTR superfamily of enzymes. Appreciably, these results offer an alternative route to produce F420 in Gram-negative model organisms and unveil yet another biochemical facet of this pathway to be explored by synthetic microbiologists.

Structural and functional impairment of surfactant protein A after exposure to nitrogen dioxide in rats

1992 ◽  
Vol 263 (2) ◽  
pp. L177-L184 ◽  
Author(s):  
B. Muller ◽  
P. Barth ◽  
P. von Wichert
Keyword(s):  
Nitrogen Dioxide ◽  
Binding Capacity ◽  
Protein A ◽  
Biological Significance ◽  
Apparent Molecular Weight ◽  
Surfactant Protein ◽  
Surfactant Protein A ◽  
In Vitro Exposure

The major surfactant protein A (SP-A) determines the dynamics of surfactant metabolism and function. We now for the first time studied the in vivo exposure of the SP-A to nitrogen dioxide (NO2) and compared it to in vitro exposure effects. Several properties that are believed to possess physiological functions were investigated. Exposure of rat SP-A to NO2 showed 1) a higher alveolar pool size after in vivo exposure, 2) a slightly decreased mannose binding capacity for the in vivo exposed form but a highly decreased binding after in vitro exposure, 3) an affected protein-lipid aggregation that was impaired most by the in vitro exposed protein, 4) a low inhibition for the secretion of phosphatidylcholine in isolated type II pneumocytes, and 5) no difference in apparent molecular weight and immunological reactivity of the differently exposed SP-A. The NO2-induced alterations of the SP-A may contribute to the pulmonary toxicity of this oxidant. It is clear from this study that the extend of the effects from in vivo and in vitro exposure are different. Therefore both forms of exposure are necessary to assess the biological significance of NO2 exposure.

scholarly journals Multisite phosphorylation of oxysterol-binding protein regulates sterol binding and activation of sphingomyelin synthesis

2012 ◽  
Vol 23 (18) ◽  
pp. 3624-3635 ◽  
Author(s):  
Asako Goto ◽  
Xinwei Liu ◽  
Carolyn-Ann Robinson ◽  
Neale D. Ridgway
Keyword(s):  
Binding Capacity ◽  
Binding Protein ◽  
Protein A ◽  
Phosphorylation Site ◽  
Oxysterol Binding Protein ◽  
Transfer Activity ◽  
Individual Site ◽  
Direct Binding ◽  
Phosphatidylinositol 4

The endoplasmic reticulum (ER)-Golgi sterol transfer activity of oxysterol-binding protein (OSBP) regulates sphingomyelin (SM) synthesis, as well as post-Golgi cholesterol efflux pathways. The phosphorylation and ER-Golgi localization of OSBP are correlated, suggesting this modification regulates the directionality and/or specificity of transfer activity. In this paper, we report that phosphorylation on two serine-rich motifs, S381-S391 (site 1) and S192, S195, S200 (site 2), specifically controls OSBP activity at the ER. A phosphomimetic of the SM/cholesterol-sensitive phosphorylation site 1 (OSBP-S5E) had increased in vitro cholesterol and 25-hydroxycholesterol–binding capacity, and cholesterol extraction from liposomes, but reduced transfer activity. Phosphatidylinositol 4-phosphate (PI(4)P) and cholesterol competed for a common binding site on OSBP; however, direct binding of PI(4)P was not affected by site 1 phosphorylation. Individual site 1 and site 2 phosphomutants supported oxysterol activation of SM synthesis in OSBP-deficient CHO cells. However, a double site1/2 mutant (OSBP-S381A/S3D) was deficient in this activity and was constitutively colocalized with vesicle-associated membrane protein–associated protein A (VAP-A) in a collapsed ER network. This study identifies phosphorylation regulation of sterol and VAP-A binding by OSBP in the ER, and PI(4)P as an alternate ligand that could be exchanged for sterol in the Golgi apparatus.

scholarly journals Developing a SARS-CoV-2 Antigen Test Using Engineered Affinity Proteins

2021 ◽  
Author(s):  
Seunghyeon Kim ◽  
Emma H. Yee ◽  
Eric A. Miller ◽  
Yining Hao ◽  
Dousabel M. Y. Tay ◽  
...  
Keyword(s):  
Infectious Diseases ◽  
Diagnostic Tests ◽  
Large Scale ◽  
Protein A ◽  
Emerging Infectious Diseases ◽  
Paper Test ◽  
N Protein ◽  
Cellulose Paper ◽  
Affinity Proteins

The ongoing COVID-19 pandemic has clearly established how vital rapid, widely accessible diagnostic tests are in controlling infectious diseases and how difficult and slow it is to scale existing technologies. Here, we demonstrate the use of the rapid affinity pair identification via directed selection (RAPIDS) method to discover multiple affinity pairs for SARS-CoV-2 nucleocapsid protein (N-protein), a biomarker of COVID-19, from in vitro libraries in 10 weeks. The pair with the highest biomarker sensitivity was then integrated into a 10-minute, vertical-flow cellulose paper test. Notably, the as-identified affinity proteins were compatible with a roll-to-roll printing process for large-scale manufacturing of tests. The test achieved 40 pM and 80 pM limits of detection in 1×PBS (mock swab) and saliva matrices spiked with cell-culture generated SARS-CoV-2 viruses and is also capable of detection of N-protein from characterized clinical swab samples. Hence, this work paves the way towards the mass production of cellulose paper-based assays which can address the shortages faced due to dependence on nitrocellulose and current manufacturing techniques. Further, the results reported herein indicate the promise of RAPIDS and engineered binder proteins for the timely and flexible development of clinically relevant diagnostic tests in response to emerging infectious diseases.

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