A Development of Anchoring Adapter Molecule for Antibody Immobilization: Monomeric Streptavidin / Antibody-binding Domain Fusion Protein

Department of Energy Engineering(Battery Science and Technology)The enzyme-linked Immunosorbent assay (ELISA) has been developed as a format of a diagnostic tool in medicine recently to detect the presence of an antigen in a liquid sample or wet sample. Especially, sandwich type ELISA has been commonly commercialized and preferred because of high specificity and sensitivity among the systems by using two antibodies to capture and detect its antigens. In addition, immobilization of antibodies is a key process to improve specificity and sensitivity in ELISA. Here we report a novel method to immobilization antibodies on the surface of the solid support with a recombinant antibody binding domain (ABD) fused with mono-streptavidin (mSA). The Z domain derived from protein A which has high binding affinity to the Fc region of various antibodies such as rabbit, rat and mouse. mSA is monomeric form of streptavidin which consists of tetramer and has very high affinity with bioitin (vitamin H). We have genetically inserted ABD with mSA (mSA-ABD) to obtain dual functions separately. mSA-ABD fusion protein is overexpressed in E.coli system and successfully purified by affinity chromatography. The binding affinity of ABD of mSA-ABD is verified with quartz crystal microbalance (QCM) and surface plasmon resonance (SPR) analysis. The other binding affinity of mSA with biotin was confirmed with binding activity test using biotin coated plates. Finally, we treat mSA-ABD as an anchoring adapter molecule in sandwich type of ELISA taking advantage of dual binding affinity and we can obtain the typical ELISA result from the series of experiments.ope

Genetically engineering encapsulin protein cage nanoparticle as a SCC-7 cell targeting optical nanoprobe

Background - Protein cage nanoparticles are promising nanoplatform candidates for efficient delivery systems of diagnostics and/or therapeutics because of their uniform size and structure as well as high biocompatibility and biodegradability. Encapsulin protein cage nanoparticle is used to develop a cell-specific targeting optical nanoprobe. Results - FcBPs are genetically inserted and successfully displayed on the surface of encapsulin to form FcBP-encapsulin. Selectively binding of FcBP-encapsulin to SCC-7 is visualized with fluorescent microscopy. Conclusions - Encapsulin protein cage nanoparticle is robust enough to maintain their structure at high temperature and easily acquires multifunctions on demand through the combination of genetic and chemical modifications.ope

TAZ stimulates exercise‐induced muscle satellite cell activation via Pard3–p38 MAPK–TAZ signalling axis

Abstract Background Exercise stimulates the activation of muscle satellite cells, which facilitate the maintenance of stem cells and their myogenic conversion during muscle regeneration. However, the underlying mechanism is not yet fully understood. This study shows that the transcriptional co‐activator with PDZ‐binding motif (TAZ) stimulates muscle regeneration via satellite cell activation. Methods Tazf/f mice were crossed with the paired box gene 7 (Pax7)creERT2 mice to generate muscle satellite cell‐specific TAZ knockout (sKO) mice. Mice were trained in an endurance exercise programme for 4 weeks. Regenerated muscles were harvested and analysed by haematoxylin and eosin staining. Muscle tissues were also analysed by immunofluorescence staining, immunoblot analysis and quantitative reverse transcription PCR (qRT‐PCR). For the in vitro study, muscle satellite cells from wild‐type and sKO mice were isolated and analysed. Mitochondrial DNA was quantified by qRT‐PCR using primers that amplify the cyclooxygenase‐2 region of mitochondrial DNA. Quiescent and activated satellite cells were stained with MitoTracker Red CMXRos to analyse mitochondria. To study the p38 mitogen‐activated protein kinase (MAPK)–TAZ signalling axis, p38 MAPK was activated by introducing the MAPK kinase 6 plasmid into satellite cells and also inhibited by treatment with the p38 MAPK inhibitor, SB203580. Results TAZ interacts with Pax7 to induce Myf5 expression and stimulates mammalian target of rapamycin signalling for satellite cell activation. In sKO mice, TAZ depletion reduces muscle satellite cell number by 38% (0.29 ± 0.073 vs. 0.18 ± 0.034, P = 0.0082) and muscle regeneration. After muscle injury, TAZ levels (2.59‐fold, P < 0.0001) increase in committed cells compared to self‐renewing cells during asymmetric satellite cell division. Mechanistically, the polarity protein Pard3 induces TAZ (2.01‐fold, P = 0.008) through p38 MAPK, demonstrating that the p38 MAPK–TAZ axis is important for muscle regeneration. Physiologically, endurance exercise training induces muscle satellite cell activation and increases muscle fibre diameter (1.33‐fold, 43.21 ± 23.59 vs. 57.68 ± 23.26 μm, P = 0.0004) with increased TAZ levels (1.76‐fold, P = 0.017). However, sKO mice had a 39% reduction in muscle satellite cell number (0.20 ± 0.03 vs. 0.12 ± 0.02, P = 0.0013) and 24% reduction in muscle fibre diameter compared to wild‐type mice (61.07 ± 23.33 vs. 46.60 ± 24.29 μm, P = 0.0006). Conclusions Our results demonstrate a novel mechanism of TAZ‐induced satellite cell activation after muscle injury and exercise, suggesting that activation of TAZ in satellite cells may ameliorate the muscle ageing phenotype and may be an important target protein for the drug development in sarcopenia

Peripheral Blood from Rheumatoid Arthritis Patients Shows Decreased Treg CD25 Expression and Reduced Frequency of Effector Treg Subpopulation

Rheumatoid arthritis (RA) is a common autoimmune disease characterized by immune cell infiltration of the synovium, leading to the loss of cartilage, bone, and joint function. Although regulatory T (Treg) cells are thought to modulate the initiation and progression of RA, a consensus has yet to be reached regarding the function and composition of Treg cells in RA patients. To address these discrepancies, we analyzed not only the total Treg frequency but also that of Treg subpopulations in the peripheral blood of RA patients and healthy controls by flow cytometry. We found that the total Treg population was not significantly different between RA and control subjects. However, the effector Treg cell subgroup, defined as CD45RA−CD25hi, showed markedly decreased frequency in RA patients. In addition, the total Treg population from RA patients showed a significant decline in the expression of CD25. Both the naïve and effector Treg subgroups also showed marked reduction of CD25 expression in RA patients compared to controls. These data suggest that the decreased frequency of effector Treg cells and overall reduction of CD25 expression in Treg cells in the peripheral blood may be evidence of altered Treg homeostasis associated with RA pathogenesis