Fc Tag Research Articles

A microfluidic electrochemical aptasensor for enrichment and detection of bisphenol A.

Bisphenol A (BPA) is an organic monomer used to make common consumer goods such as plastic containers, sports equipment, and cosmetics which are heavily produced worldwide. A growing interest has been drawn to general public as BPA is one of the major endocrine disrupting chemicals threating human health. To date, numerous BPA sensors have been attempted to be developed but important challenges still remained such as limited linearity range, easy to use, and long term response time. To address the present issues, a microfluidic channel should be integrated into an electrochemical aptasensor and it is called Geometrically Activated Surface Interaction (GASI) chip. The vigorous generation of the micro-vortex in the GASI fluidic chamber provides the high collision chances between BPA and anti-BPA aptamer (BPAPT) and consequently more BPA molecules can be captured on the aptasensor surface, which finally results in high sensitivity of the aptasensor. To construct the integrated aptasensor, a miniaturized gold electrode is fabricated using shadow mask and e-beam evaporation process. Afterward, BPAPT is immobilized on a nanostructured gold electrode via thiol chemistry, and other terminus of the aptamer is labeled with a ferrocene (Fc) redox probe. Then, the microfluidic channel is mounted over the miniaturized gold electrode to introduce and enrich BPA to the aptasensor. Upon the specific interaction between BPA and its aptamer, configuration of aptamer is changed so that Fc tag approaches to the electrode surface and direct oxidation signal of Fc and BPA are followed as analytical signals. The unique microfluidic integrated electrochemical aptasensor delivers a wide linear dynamic range over 5 × 10–12 to 1 × 10−9 M, with a limit of detection 2 × 10–13 M. This aptasensor provides a precise platform for simple, selective and more importantly rapid detection of BPA. Such kind of sensing platforms can serve as a fertile ground for designing miniaturized portable sensors.

Native Folding of a Recombinant gpE1/gpE2 Heterodimer Vaccine Antigen from a Precursor Protein Fused with Fc IgG.

A prophylactic HCV vaccine is still needed to control this global disease despite the availability of direct-acting antivirals. Previously, we demonstrated that a recombinant envelope glycoprotein (E1E2) vaccine (genotype 1a) elicited cross-neutralizing antibodies from human volunteers. A challenge for isolating the E1E2 antigen is the reliance on GNA, which is unsuitable for large scale-up and global vaccine delivery. We have generated a novel Fc domain-tagged E1E2 antigen that forms functional heterodimers similar to those with native E1E2. Affinity purification and removal of the Fc tag from E1E2 resulted in an antigen with a nearly identical profile of cross-neutralizing epitopes. This antigen elicited anti-HCV antibodies that targeted conserved neutralizing epitopes of E1E2. Owing to the high selectivity and cost-effective binding capacity of affinity resins for capture of the Fc-tagged rE1E2, we anticipate that our method will provide a means for large-scale production of this HCV vaccine candidate.

Maltose-Binding Protein (MBP), a Secretion-Enhancing Tag for Mammalian Protein Expression Systems.

Recombinant proteins are commonly expressed in eukaryotic expression systems to ensure the formation of disulfide bridges and proper glycosylation. Although many proteins can be expressed easily, some proteins, sub-domains, and mutant protein versions can cause problems. Here, we investigated expression levels of recombinant extracellular, intracellular as well as transmembrane proteins tethered to different polypeptides in mammalian cell lines. Strikingly, fusion of proteins to the prokaryotic maltose-binding protein (MBP) generally enhanced protein production. MBP fusion proteins consistently exhibited the most robust increase in protein production in comparison to commonly used tags, e.g., the Fc, Glutathione S-transferase (GST), SlyD, and serum albumin (ser alb) tag. Moreover, proteins tethered to MBP revealed reduced numbers of dying cells upon transient transfection. In contrast to the Fc tag, MBP is a stable monomer and does not promote protein aggregation. Therefore, the MBP tag does not induce artificial dimerization of tethered proteins and provides a beneficial fusion tag for binding as well as cell adhesion studies. Using MBP we were able to secret a disease causing laminin β2 mutant protein (congenital nephrotic syndrome), which is normally retained in the endoplasmic reticulum. In summary, this study establishes MBP as a versatile expression tag for protein production in eukaryotic expression systems.

Highly sensitive electrochemical detection of mercury (II) via single ion-induced three-way junction of DNA

Abstract A “signal-on” electrochemical sensing strategy was designed for highly sensitive and selective detection of mercury (II) via its induction to three-way junction of DNA (DNA-TWJ). The TWJ consisted of the capture probe that was self-assembled on a gold electrode surface through S Au bond, the signal probe that was labeled with ferrocene (Fc) and contained single T–T mismatch to capture probe, and an assistant probe for the formation of DNA-TWJ upon the presence of mercury (II). This process caused the Fc tag approaching the electrode for fast electron transfer and thus increased the oxidation current. The “signal-on” sensing method could detect Hg 2 + ranging from 0.005 to 100 nM. The assay was simple and fast. It showed potential application in on-site and real-time Hg 2 + detection.

Anti-RANKL treatment improves screw fixation in cancellous bone in rats

Bisphosphonates improve implant fixation in randomised clinical trials of knee prostheses, hip prostheses and dental implants. However, a limited amount of bone resorption is required for bisphosphonates to exert an effect. Anti-RANKL treatment does not have this limitation, and we therefore tested whether if they might be more effective for improvement of implant fixation. This is of interest, as anti-RANKL treatment with denosumab is now in common clinical use.Male SD rats received a stain-less steel screw in the right proximal tibia and a drill hole in the left (n=42). They were randomised to subcutaneous injections of either alendronate (20μg/kg/day), alendronate (200μg/kg/day), osteoprotegerin with an Fc tag (OPG-Fc; 8mg/kg, twice weekly), or saline control. After 4 weeks, the fixation of the steel screw was measured by pull-out test. The tibia with the drill hole was evaluated with μCT.OPG-Fc increased the pull-out force compared to saline controls by 153% (p<0.001). There was no significant difference between OPG-Fc and the alendronate groups.OPG-Fc increased the bone density (BV/TV) in the previous drill hole compared to controls 7-fold (p<0.001). This increase was higher than with any alendronate dose (p<0.001).OPG-Fc increased the bone density of the L5 vertebral body, but there was no significant difference between OPG-Fc and alendronate.Our results suggest that screw fixation in cancellous bone can be dramatically improved by an anti-RANKL agent. The effect was comparable to very high bisphosphonate doses. Screw insertion in cancellous bone elicits a metaphyseal fracture healing response, and our findings might be relevant not only for implant fixation, but also for fracture healing in cancellous bone.

Abstract 2661: Patient-derived breast cancer targeting heavy chain antibodies

Abstract The emergence of tumor-targeting antibodies such as Trastuzumab, Rituximab and anti-CD47 antibody has highlighted the significant role of antibodies for effective cancer therapy. The most common approach to generate tumor specific antibodies is to select an established tumor specific antigen and generate antibodies to that target. The drawback with this approach is that it does not lead to identification of novel antigens or antigenic epitopes. Therefore, we developed a strategy that will lead to the identification of both tumor specific antibodies and novel tumor antigens. The approach uses the patient's immune system to guide us to define tumor specific antigens. In order to achieve this, we identified reactive germinal centers in the tumor draining sentinel lymph nodes from breast cancer patients. Immunohistochemical analysis of reactive germinal centers defined the B-cell regions for mRNA isolation and generation of cDNA molecules for the variable heavy chain segment (VH) of antibodies. The VH cDNA molecules were cloned into a mammalian expression vector to generate bivalent heavy chain antibodies with a C-terminus mouse Fc tag. At present, we have generated about 250 antigen-driven variable heavy chain clones from 27 breast cancer patient lymph node samples. We screened 46 heavy chain antibodies against a panel of breast cancer cell lines using immunofluorescence and identified 8 antibodies that showed strong and specific staining. Of these 8 antibodies, one of them (VH6-93) showed membrane staining preferentially on MDA-MB-231 cells in comparison to MCF7 cells. VH6-93 antibody also showed strong staining on conditionally reprogrammed tumor cells derived from a breast cancer patient with only moderate staining on normal breast epithelial cells derived from the same patient. VH6-93 also revealed preferential binding to breast tumor tissues as compared to normal ductal tissues evidenced by immunofluorescence and immunohistochemistry. To identify the target antigens, a multi-tiered approach was used, which included immunoprecipitation of target antigen from cell lysates followed by peptide sequencing with mass spectrometry as well as utilizing Nucleic Acid Programmable Protein Array (NAPPA) technology. A comprehensive analysis of two patient-derived antibodies has produced a panel of potential antigen targets, which are undergoing specificity and validation analysis. In summary, this methodology of using activated B-cells from sentinel lymph nodes is a unique way to enrich for tumor-specific antibody clones. The findings from this research will not only lead to tumor specific antibodies but will also reveal novel tumor specific antigens. Identification of these novel antigens can give rise to additional therapeutic strategies such as peptide or protein vaccination. Citation Format: Charan Kumar V. Devarakonda, Daniel Kita, Kathryn N. Phoenix, Dewey M. Magee, Kevin P. Claffey. Patient-derived breast cancer targeting heavy chain antibodies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2661. doi:10.1158/1538-7445.AM2014-2661

Searching for an ideal vaccine candidate among different MERS coronavirus receptor-binding fragments—The importance of immunofocusing in subunit vaccine design

The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) is currently spreading among humans, making development of effective MERS vaccines a high priority. A defined receptor-binding domain (RBD) in MERS-CoV spike protein can potentially serve as a subunit vaccine candidate against MERS-CoV infections. To identify an ideal vaccine candidate, we have constructed five different versions of RBD fragments, S350-588-Fc, S358-588-Fc, S367-588-Fc, S367-606-Fc, and S377-588-Fc (their names indicate their residue range in the spike protein and their C-terminal Fc tag), and further investigated their receptor binding affinity, antigenicity, immunogenicity, and neutralizing potential. The results showed that S377-588-Fc is among the RBD fragments that demonstrated the highest DPP4-binding affinity and induced the highest-titer IgG antibodies in mice. In addition, S377-588-Fc elicited higher-titer neutralizing antibodies than all the other RBD fragments in mice, and also induced high-titer neutralizing antibodies in immunized rabbits. Structural analysis suggests that S377-588-Fc contains the stably folded RBD structure, the full receptor-binding site, and major neutralizing epitopes, such that additional structures to this fragment introduce non-neutralizing epitopes and may also alter the tertiary structure of the RBD. Taken together, our data suggest that the RBD fragment encompassing spike residues 377-588 is a critical neutralizing receptor-binding fragment and an ideal candidate for development of effective MERS vaccines, and that adding non-neutralizing structures to this RBD fragment diminishes its neutralizing potential. Therefore, in viral vaccine design, it is important to identify the most stable and neutralizing viral RBD fragment, while eliminating unnecessary and non-neutralizing structures, as a means of “immunofocusing”.

Binding-induced internal-displacement of inverted aptamer beacon: Toward a novel electrochemical detection platform

In this paper, we reported a novel electrochemical aptasensor based on an inverted aptamer beacon (IAB). The IAB architecture with a hairpin structure consisted of an aptamer arm and a ferrocene (Fc)-labeling signal arm, and was inverted on the electrode through a thiol-linker on the middle site of its loop. The inverted immobilization strategy prevented the end-labeling of ferrocene tag from approaching the electrode, thus resulting in a weak electrochemical signal. In the presence of thrombin, binding of target with the aptamer arm displaced the signaling arm, producing a flexible single-stranded element, i.e. internal-displacement which allowed Fc tag to collide with the electrode, thereby producing a relatively strong electrochemical signal. By monitoring the increased signal, thrombin could be readily detected in the signal-on mode with a detection limit (LOD) of 0.21nM. Inspiringly, the IAB-based strategy required no auxiliary strands and did not depend on structure-switching of specific aptamer/target couple, thus presenting as a generalized method for fabrication of other electrochemical aptasensors.

Immune response to Fc tagged GP5 glycoproteins of porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) glycoprotein 5 (GP5) is the most abundant envelope glycoprotein and a key target for neutralizing antibodies. Previous studies have demonstrated that the native GP5 glycoprotein is poorly immunogenic and not able to induce robust protective responses, probably due to the presence of a non-neutralizing decoy epitope and shielding N-linked glycans close to its neutralizing epitope. In the present report, two Fc tagged GP5 proteins (GP5-Fc containing a truncated GP5 with the deletion of its signal peptide and transmembrane regions, and GP5N-Fc containing only the ectodomain of GP5) were designed based on the sequences of a highly pathogenic PRRSV strain and produced using a baculovirus/insect cell expression system. Immunization studies showed that both GP5-Fc and GP5N-Fc elicited strong serum responses in the absence of adjuvant to the native GP5 present on the surface of purified PRRSV virons. Although GP5N-Fc failed in inducing significant titer of neutralizing antibodies in mice, GP5-Fc was shown as an effective inducer of neutralizing antibodies specific to PRRSV. Our results suggest that the modified GP5 glycoprotein with Fc tag has the potential to be a candidate for the future development of new generation vaccine against PRRSV infection.

Enzyme-free amplification for sensitive electrochemical detection of DNA via target-catalyzed hairpin assembly assisted current change

An isothermal, enzyme-free and sensitive method for electrochemical detection of DNA is proposed based on target catalyzed hairpin assembly and for signal amplification. Molecular beacon 1 (MB1) contains a ferrocene (Fc) tag, which was immobilized on the gold electrode as recognition probe to hybridize with target DNA. Then, molecular beacon 2 hybridized with the opened MB1, allowing the target to be displaced. The displaced target again triggered the next round of strand exchange reaction resulting in many Fc far away from the GE to achieve signal amplification for sensitive DNA detection. The current signal amplification strategy is relatively simple and inexpensive owing to avoid the use of any kind of enzyme or sophisticated equipment. It can achieve a sensitivity of 42fM with a wide linear dynamic range from 10−13 to 10−9M and discriminate mismatched DNA from perfect matched target DNA with a high selectivity. The proposed method showed excellent specificity, high sensitivity and low detection limit, and could be applied in analysis of real samples.

Nonmuscle Myosin Heavy Chain IIA Is a Critical Factor Contributing to the Efficiency of Early Infection of Severe Fever with Thrombocytopenia Syndrome Virus

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a novel phlebovirus in the Bunyaviridae family. Most patients infected by SFTSV present with fever and thrombocytopenia, and up to 30% die due to multiple-organ dysfunction. The mechanisms by which SFTSV enters multiple cell types are unknown. SFTSV contains two species of envelope glycoproteins, Gn (44.2 kDa) and Gc (56 kDa), both of which are encoded by the M segment and are cleaved from a precursor polypeptide (about 116 kDa) in the endoplasmic reticulum (ER). Gn fused with an immunoglobulin Fc tag at its C terminus (Gn-Fc) bound to multiple cells susceptible to the infection of SFTSV and blocked viral infection of human umbilical vein endothelial cells (HUVECs). Immunoprecipitation assays following mass spectrometry analysis showed that Gn binds to nonmuscle myosin heavy chain IIA (NMMHC-IIA), a cellular protein with surface expression in multiple cell types. Small interfering RNA (siRNA) knockdown of NMMHC-IIA, but not the closely related NMMHC-IIB or NMMHC-IIC, reduced SFTSV infection, and NMMHC-IIA specific antibody blocked infection by SFTSV but not other control viruses. Overexpression of NMMHC-IIA in HeLa cells, which show limited susceptivity to SFTSV, markedly enhanced SFTSV infection of the cells. These results show that NMMHC-IIA is critical for the cellular entry of SFTSV. As NMMHC-IIA is essential for the normal functions of platelets and human vascular endothelial cells, it is conceivable that NMMHC-IIA directly contributes to the pathogenesis of SFTSV and may be a useful target for antiviral interventions against the viral infection.

Sushi domain‐containing protein 4 (SUSD4) inhibits complement by disrupting the formation of the classical C3 convertase

Recently discovered Sushi domain-containing protein 4 (SUSD4) contains several Sushi or complement control protein domains; therefore, we hypothesized that it may act as complement inhibitor. Two isoforms of human SUSD4, fused to the Fc part of human IgG, were recombinantly expressed in Chinese hamster ovary (CHO) cells. The secreted soluble isoform of SUSD4 (SUSD4b) inhibited the classical and lectin complement pathways by 50% at a concentration of 0.5 μM. This effect was due to the fact that 1 μM SUSD4b inhibited the formation of the classical C3 convertase by 90%. The membrane-bound isoform (SUSD4a) inhibited the classical and alternative complement pathways when expressed on the surface of CHO cells but not when expressed as a soluble, truncated protein. In all functional studies, we used known complement inhibitors as positive controls, while Coxsackie adenovirus receptor, which has no effect on complement, expressed with Fc tag, was a negative control. We also studied the mRNA expression of both isoforms of SUSD4 in a panel of human tissues using quantitative PCR and primarily found SUSD4a in esophagus and brain, while SUSD4b was highly expressed in esophagus, ovary, and heart. Overall, our results show that SUSD4 is a novel complement inhibitor with restricted expression.

Identification of Active Sequences in the L4a Domain of Laminin α5 Promoting Neurite Elongation

Laminin α5 is an extracellular matrix protein containing multiple domains implicated in various biological processes, such as embryogenesis and renal function. In this study, we used recombinant proteins and synthetic peptides to identify amino acid residues within the short arm region of α5 that were critical for neurite outgrowth activity. The short arm of α5 contains three globular domains (LN, L4a, and L4b) and three rodlike elements (LEa, LEb, and LEc). Recombinant proteins comprised of the α5 short arm fused with a Fc tag produced in 293 cells were assayed for PC12 (pheochromocytoma) cell adhesion and neurite outgrowth activities. Although it did not have cell attachment activity, neurite outgrowth was promoted by the recombinant protein. To narrow the region involved in neurite outgrowth activity, two truncated recombinant proteins were produced in 293 cells. A recombinant protein lacking L4a and LEb lost activity. Furthermore, we synthesized 78 partially overlapping peptides representing most of the amino acid sequences of L4a and LEb. Of the peptides, A5-76 [mouse laminin α5 928-939 (TSPDLFRLVFRY) in L4a] exhibited neurite outgrowth activity. Mutagenesis studies showed that Phe(933) and Arg(934) were involved in neurite outgrowth activity. Moreover, inhibition assays using anti-integrin monoclonal antibodies showed that neurite outgrowth on the α5 short arm was partially mediated by integrin α1β1. However, the antibodies to integrin α1 and β1 did not inhibit neurite elongation on the A5-76 peptide. These results suggest that in addition to cellular interactions with the active site in the L4a domain, the binding of integrin α1β1 seems to modulate neurite elongation on the short arm of α5.

Pregnancy-specific Glycoprotein 1 Induces Endothelial Tubulogenesis through Interaction with Cell Surface Proteoglycans

Pregnancy-specific β1 glycoproteins (PSGs) are the most abundant fetal proteins in the maternal bloodstream in late pregnancy. They are secreted by the syncytiotrophoblast and are detected around day 14 postfertilization. There are 11 human PSG genes, which encode a family of proteins exhibiting significant conservation at the amino acid level. We and others have proposed that PSGs have an immune modulatory function. In addition, we recently postulated that they are proangiogenic due to their ability to induce the secretion of VEGF-A and the formation of tubes by endothelial cells. The cellular receptor(s) for human PSGs remain unknown. Therefore, we conducted these studies to identify the receptor for PSG1, the highest expressed member of the family. We show that removal of cell surface glycosaminoglycans (GAGs) by enzymatic or chemical treatment of cells or competition with heparin completely inhibited binding of PSG1. In addition, PSG1 did not bind to cells lacking heparan or chondroitin sulfate on their surface, and binding was restored upon transfection with all four syndecans and glypican-1. Importantly, the presence of GAGs on the surface of endothelial cells was required for the ability of PSG1 to induce tube formation. This finding indicates that the PSG1-GAG interaction mediates at least some of the PSG1 proposed functions.

Crystal Structure of CD14 and Its Implications for Lipopolysaccharide Signaling

Lipopolysaccharide, the endotoxin of Gram-negative bacteria, induces extensive immune responses that can lead to fatal septic shock syndrome. The core receptors recognizing lipopolysaccharide are CD14, TLR4, and MD-2. CD14 binds to lipopolysaccharide and presents it to the TLR4/MD-2 complex, which initiates intracellular signaling. In addition to lipopolysaccharide, CD14 is capable of recognizing a few other microbial and cellular products. Here, we present the first crystal structure of CD14 to 2.5 angstroms resolution. A large hydrophobic pocket was found on the NH2-terminal side of the horseshoe-like structure. Previously identified regions involved in lipopolysaccharide binding map to the rim and bottom of the pocket indicating that the pocket is the main component of the lipopolysaccharide-binding site. Mutations that interfere with lipopolysaccharide signaling but not with lipopolysaccharide binding are also clustered in a separate area near the pocket. Ligand diversity of CD14 could be explained by the generous size of the pocket, the considerable flexibility of the rim of the pocket, and the multiplicity of grooves available for ligand binding.

Alkaline phosphatase fusion proteins for molecular characterization and cloning of receptors and their ligands

A large number of so-called orphan receptors have been identified that appear by their structure to be cell surface receptors for polypeptide ligands, yet have no known ligand. This has implied an opportunity to identify corresponding ligands that would be novel cell-cell signaling molecules. One strategy to identify such molecules has been to use the extracellular domain of the orphan receptor, fused to an alkaline phosphatase (AP) or immunoglobulin Fc tag that can serve as an affinity probe to identify the corresponding ligand(s). This approach is widely applicable, and has been used to identify a large number of novel ligands. An analogous strategy, using ligands fused to AP or Fc tags, has similarly been used to identify novel receptors. The chapter describes the use of such fusion proteins to identify and clone novel ligands or receptors. It also describes methods for the molecular characterization of ligands, receptors, and their interactions. An efficient procedure to identify a good source for cloning is to test embryonic or adult tissues by in situ staining, using a receptor or ligand-AP fusion protein as a probe to identify locations of high expression.