• Journal of Sensor Science and Technology
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• v.19 no.2
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• pp.142-148
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• 2010

We have compared and investigated the detection capabilities of antibody of immunoglobulin G(anti-IgG) immobilized by protein G and N-hydroxysuccinimide(NHS) at the end of the self-assembled monolayer(SAM). Surface plasmon resonance(SPR) sensor has been utilized to measure the interaction between biomolecules. After formation of the protein G and SAM, anti-IgG, bovine serum albumin(BSA) and IgG has been sequently injected. Through the reponse of the SPR, we can conclude that the protein G immobilized anti-IgG better than the SAM. In addition, IgG detection capability of the anti-IgG immobilized by the protein G showed better performance compared with that immobilized by the SAM.

• Korean Chemical Engineering Research
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• v.58 no.2
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• pp.280-285
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• 2020

We have developed a constitutive display system of the Pseudomonas fluorescens SIK W1 TliA lipase on the cell surface of Escherichia coli using E. coli outer membrane protein C (OmpC) as an anchoring motif, which is an economical compared to induced system. For the constitutive expression of truncated OmpC-TliA fusion proteins, gntT104 promoter was employed. Cell growth was not affected by over expression of fusion protein during entire culture time, suggesting cell lysis was not a problem. The localization of truncated OmpC-TliA fusion protein on the cell surface was confirmed by immunofluorescence microscopy and measuring whole cell lipase activity. Constitutively displayed lipase was very stable, retaining activity enantioselectivity throughout the five repeated reactions. These results suggest that OmpC from E. coli be a useful anchoring motif for displaying enzymes on the cell surface without any inducers, and this stable surface display system can be employed for a broad range of biotechnological applications.

• Macromolecular Research
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• v.9 no.4
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• pp.197-205
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• 2001

Platelet adhesion onto elastic polymeric biomaterials was tested in vitro by perfusing human whole blood at a shear rate of 100 sec$\^$-1/ for possible verification of mechanisms of initial platelet adhesion perfusion of blood on the polymeric substrates was performed after treatments either with or without pre-adsorption of 1% blood plasma, and either with or without residence of the protein-preadsorbed substrate in phosphate buffered solution. The surfaces employed were elastic polymers such as poly(ether urethane urea), poly(ether urethane), silicone urethane copolymer, silicone rubber and poly(ether urethane) with the anti-calcifying agent hydroxyethane bisphosphate. Each polymer surface treated was exposed in vitro to the dynamic, heparinized whole blood perfused for upto 6 min and the surface area of platelets initially adhered was measured by employing in situ epifluorescence video microscopy. The blood perfusion was performed on the surfaces treated at the following three different conditions: directly on the bare surfaces, after protein pre-adsorption and after residence in buffer for 3 days of the surfaces protein pre-adsorbed for 2 h. The effects of blood plasma pre-adsorption on the initial platelet adhesion was surface-dependent. The amount of the adsorbed fibrinogen and the surface coverage area of the adhered platelets were dependent on the surface conditions whether substrates were bare surfaces or protein pre-adsorbed ones. To test an effect of possible morphological (re)orientations of the adsorbed proteins on the initial platelet adhesion, the polymeric substrate pre-adsorbed with 1% blood plasma was immersed in phosphate buffered solution for 3 days and then exposed to physiological blood perfusion. The surface area of the platelets adhered on these surfaces was significantly different from that of the surfaces treated with protein pre-adsorption only. These results indicated that platelet adhesion was dependent on the surface property itself and pre-treatment conditions such as blood perfusion without any pre-adsorption of proteins, and blood perfusion either after protein pre-adsorption or after subsequent substrate residence in buffer of the substrate pre-adsorbed with proteins. Understanding of these results may guide for better designs of blood-contacting materials based on protein behaviors.

• BMB Reports
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• v.28 no.6
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• pp.504-508
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• 1995

The electrophoretic patterns of plasma membrane surface proteins of normal rat liver cells and rat hepatomas were compared in 10% non-denaturing and 7-15% gradient non-denaturing gel. Chemical carcinogens, 2-Me DAB (2-methyl-4-dimethylaminoazobenzene) and DENA (diethylnitrosamine), were used to induce hepatoma in rats. One protein which disappeared in hepatoma was identified in normal rat liver by non-denaturing gel electrophoresis. Rabbit antisera were raised against this specific protein, and the protein was purified by Sephacryl S-200 column and immunoaffinity chromatography using the purified antibody. The purified protein showed two bands of molecular weights approximately 50 $kD_{\alpha}$ and 52 $kD_{\alpha}$ by SDS-polyacrylamide gel electrophoresis, which reacted specifically with the antibody. However only one band was observed in non-denaturing gel and also in isoelectric focusing with a pI value of 6.6. This study showed the existence of an unique protein on the plasma membrane surface of normal rat liver cells which disappeared in rat hepatomas induced by chemical carcinogens.

• Biotechnology and Bioprocess Engineering:BBE
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• v.9 no.2
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• pp.137-142
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• 2004

In this study, a simple procedure is described for patterning biotin on a glass substrate and then selectively immobilizing proteins of interest onto the biotin-patterned surface. Microcontact printing (CP) was used to generate the micropattern of biotin and to demonstrate the selective immobilization of proteins by using enhanced green fluorescent protein (EGFP) as a model protein, of which the C-terminus was fused to a core streptavidin (cSA) gene of Streptomyces avidinii. Confocal fluorescence microscopy was used to visualize the pattern of the immobilized protein (EGFP-cSA), and surface plasmon resonance was used to characterize biological activity of the immobilized EGFP-cSA. The results suggest that this strategy, which consists of a combination of $\mu$CP and cSA-fused proteins. is an effective way for fabricating biologically active substrates that are suitable for a wide variety of applications. one such being the use in protein-protein assays.

• Biomaterials and Biomechanics in Bioengineering
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• v.3 no.1
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• pp.59-69
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• 2016

A surface resisting protein adsorption and cell adhesion is highly desirable for many biomedical applications such as diagnostic devices, biosensors and blood-contacting devices. In this study, a surface conjugated with sulfobetaine molecules was fabricated via the click reaction for the anti-fouling purpose. An alkyne-containing substrate (Alkyne-PPX) was generated by chemical vapor deposition of 4-ethynyl-[2,2]paracyclophane. Azide-ended mono-sulfobetaine molecules were synthesized and then conjugated on Alkyne-PPX via the click reaction. The protein adsorption from 10% serum was reduced by 57%, while the attachment of L929 cells was reduced by 83% onto the sulfobetaine-PPX surface compared to the protein adsorption and cell adhesion on Alkyne-PPX. In conclusion, we demonstrate that conjugation of mono-sulfobetaine molecules via the click chemistry is an effective way for reduction of non-specific protein adsorption and cell attachment.

• Biotechnology and Bioprocess Engineering:BBE
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• v.5 no.6
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• pp.460-464
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• 2000

Extracellular polymeric substances (EPS) are believed to play a role in the binding and formation of microbial flocs. However, the precise role is not well known. Sludge settling characteristics and the carbohydrate to protein ratio in EPS were tested with various airflow rates in this study. Sludge was collected from three modified sequencing batch reactors (SBRs), which were operated at 16$\^{C}$ with an airflow rate of 0.8L/min, 3L/min and 6L/min, respectively. During the operation, the reactor operated at an airflow rate of 0.8L/min showed sludge volume index (SVI) of 80 to 90ml/g and a constant ratio of carbohydrate to protein in the EPS, while a significant increase in the SVI was seen in the other reactors. Sludge bulking increased the amount of carbohydrate in the EPS, while kept protein almost constant in the airflow rate of 3L/min ad 6L/min. Surface charge also increased with increases in the carbohydrate to protein ratio in the EPS, which weakens the attraction between the EPS and multivalent cations. The ratio of carbohydrate to protein in the EPS was tween the EPS and multivalent cations. The ratio of carbohydrate to protein in the EPS was inferred to be essential for bioflocculation.

• BMB Reports
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• v.29 no.2
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• pp.180-182
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• 1996

One of the essential functions of virus surface proteins is the recognition of specific receptors on target cell membranes, and cellular receptors play an important role in viral pathogenesis. But the earliest steps of hepatitis B virus (HBV) infection, such as hepatocyte receptor interaction with the virus, are poorly understood. Previous work has suggested an important role of the preS1 region of HBV envelope protein in mediating viral binding to hepatocytes. Although hepatitis B virus (HBV) infection appears to be initiated by specific binding of virions to cell membrane structures via one or potentially several viral surface proteins, data showing the identification or isolation of the HBV receptor (s) are not yet available. The receptor-like proteins on the plasma membrane surface of HepG2 cells that bind to PreS1 were separated and identified using affinity chromatography, and the amino-terminal amino acid sequences of the receptor-like proteins were determined.

• Biotechnology and Bioprocess Engineering:BBE
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• v.8 no.4
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• pp.227-232
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• 2003

A self-assembled monolayer of protein G was fabricated to develop an immunosensor based on surface plasmon resonance (SPR), thereby improving the performance of the antibodybased biosensor through immobilizing the antibody molecules (lgG). As such, 11-mercaptoundecanoic acid (11-MUA) was adsorbed on a gold (Au) support, while the non-reactive hydrophilic surface was changed through substituting the carboxylic acid group (-COOH) in the 11-MUA molecule using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrocholide (EDAC). The formation of the self-assembled protein G layer on the Au substrate and binding of the antibody and antigen were investigated using SPR spectroscopy, while the surface topographies of the fabricated thin films were analyzed using atomic force microscopy (AFM). A fabricated monoclonal antibody (Mab) layer was applied for detecting E. coli O157:H7. As a result, a linear relationship was achieved between the pathogen concentration and the SPR angle shift, plus the detection limit was enhanced up to 10$^2$ CFU/mL.

• Journal of Microbiology and Biotechnology
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• v.16 no.8
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• pp.1169-1173
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• 2006

Imaging ellipsometry (IE) for detection of binding of Escherichia coli O157:H7 (E. coli O157:H7) to an immunosensor is reported. A protein G layer, chemically bound to a self-assembled layer of 11-mercaptoundecanoic acid (11-MUA), was adopted for immobilization of monoclonal antibody against E. coli O157:H7 (Mab). The immobilization of antibody was investigated using surface plasmon resonance. To fabricate antibody spots on a gold surface, protein G solution was spotted onto the gold surface modified with an 11-MUA layer, followed by immobilizing Mab on the protein G spot. Ellipsometric images of the protein G spot, the Mab spot, and Mab spots with binding of E. coli O157:H7 in various concentrations were acquired using the IE system. The change of mean optical intensity of the Mab spots in the ellipsometric images indicated that the lowest detection limit was $10^3$CFU/ml for E. coli O157:H7. Thus, IE can be applied to an immunosensor for detection of E. coli O157:H7 as a detection method with the advantages of allowing label-free detection, high sensitivity, and operational simplicity.