• KSBB Journal
• /
• v.21 no.4
• /
• pp.279-285
• /
• 2006

We investigated the effects of immobilization chemistry on the yield of immobilization and the bioactivity of the immobilized enzymes. Trypsin as a model protein and macroporous polymer beads(Toyopearl AF 650M, Tosho Co., Japan) was used as a model matrix. Four methods were used to immobilize trypsin; covalent conjugation by reductive amination(at pH 10.0 and pH 4.0) and affinity interaction via streptavidin-biotin, and double-affinity interaction via biotin-streptavidin-biotin system. The covalent conjugation immobilized $3{\sim}4$ mg/ml-gel, ca. 3-fold higher than the affinity method. However, the specific activity of the covalently(pH 10.0) and affinity-immobilized trypsin(via streptavidin-biotin) are ca. 37% and 50%, respectively, of that of the soluble enzyme(on the low-molecular-weight BAPNA substrate). When the molecular size of a substrate increased, the affinity-immobilized trypsin showed higher clavage activity on insulin and BSA. This result seemed to indicate the streptavidin-biotin system allowed more steric flexibility of the immobilized trypsin in its interaction with a substrate molecule. To confirm this, we studied the molecular flexibility of immobilized trypsin using quartz crystal microbalance-dissipation. Self-assembled monolayers were formed on the Q-sensor surface by aminoalkanethiols, and gultaraldehyde was attached to the SAMs. Trypsin was immobilized in two ways: reductive amination(at pH 10.0) and the streptavidin-biotin system. The dissipation shift of the affinity-immobilized trypsin was $0.8{\times}10^{-6}$, whereas that of the covalently attached enzyme was almost zero. This result confirmed that the streptavidin-biotin system allowed higher molecular flexibility. These results suggested that the bioactivity of the immobilized enzyme be strongly dependent on its molecular flexibility.

• Journal of Life Science
• /
• v.25 no.6
• /
• pp.631-637
• /
• 2015

Streptavidin, a protein produced by Streptomyces avidinii, strongly binds up to four molecules of vitamin H, d-biotin exhibiting the dissociation constant of about 10⁻¹⁵ M. This strong binding affinity has been applied for detection and characterization of numerous biological molecules suggesting expression and purification of functional streptavidin should be very useful for the application of this streptavidin-biotin interaction. To express a soluble streptavidin in Escherichia coli, We synthesized streptavidin genes and cloned into pET-22b plasmid, which uses T7 RNA polymerase/T7 promoter expression systems containing pelB leader for secretion into periplasmic space and six polyhistidine tags at C-terminus for purification of expressed proteins. Although streptavidin is toxic to Escherichia coli due to strong biotin binding property, streptavidin was expressed very sufficiently in a range of 10-20 mg/ml. In SDS-PAGE, the size of purified protein was shown as 17 kDa in denatured condition (boiling) and 68 kDa in native condition (without boiling) suggesting tetramerization of monomeric subunit by non-covalent association. Further analysis by size-exclusion chromatography supported streptavidin’s tetrameric structure as well. In addition, soluble streptavidin detected biotinylated proteins in westernblot indicating its functional activity to biotin. Taken these results together, it concluded that our simple expression system was able to show high yield, homotetrameric formation and biotin binding activity analogous to natural streptavidin.

• Korean Journal of Microbiology
• /
• v.54 no.4
• /
• pp.330-342
• /
• 2018

The Streptavidin and Biotin system has been studied most extensively as the high affinity non-covalent binding of Biotin to STR ($K_D=10^{-14}M$) and four Biotin binding sites in tetrameric Streptavidin makes this system useful for the production of multivalent antibody. For the application of this system, we cloned Streptavidin amplified from Streptomyces avidinii chromosome by PCR and fused to gene of hAY4 single-chain Fv antibody specific to death receptor 4 (DR4) which is a receptor for tumor necrosis factor ${\alpha}$ related apoptosis induced ligand. The hAY4 single-chain Fv antibody fused to Streptavidin expressed in Escherichia coli showed 43 kDa monomer in heated SDS-PAGE. However, this fusion protein shown in both non-heated SDS-PAGE and Size-exclusion chromatography exhibited 172 kDa as a tetramer suggesting that natural tetramerization of Streptavidin by non-covalent association induced hAY4 single-chain Fv tetramerization. This fusion protein retained a Biotin binding activity similar to natural Streptavidin as shown in Ouchterlony assay and ELISA. Death receptor 4 antigen binding activity of purified hAY4 single-chain Fv fused to Streptavidin was also confirmed by ELISA and Westernblot. In addition, surface plasmon resonance analysis showed 60-fold higher antigen binding affinity of the hAY4-STR than monomeric hAY4 ScFv due to tetramerization. In summary, hAY4 single-chain Fv fused to Streptavidin fusion protein was successfully expressed and purified as a soluble tetramer in E. coli and showed both Biotin and DR4 antigen binding activity suggesting possible production of bifunctional and tetrameric ScFv antibody.

• Journal of Sensor Science and Technology
• /
• v.4 no.4
• /
• pp.81-87
• /
• 1995

A novel biomaterial capable of incorporating biotinylated biomolecule has been synthesized. Our strategy is to biotinylate one-dimensional electroactive polymers and use a bridging streptavidin protein on Langmuir-Blodgett (LB) organized films. These copolymers are derivatized with long alkyl chains and biotin moieties to bind, respectively, to the hydrophobic surface and the biotinylated species, through the biotin and streptavidin complexation. We utilize the polymer assembly approach to attach a signal transducing biomolecule biotinylated phycoerythrin (B-PE) into this novel biomaterial by binding the unoccupied biotin binding sites on the bound streptavidin (4 sites total). The pressure-area isotherm of the protein injected monolayer showed area expansion. A characteristic fluorescent emission peak at 576nm was detected from the monolayer transferred onto a solid substrate. These observations demonstrated the promise of the organized thin polymer assemblies for their application to the sensor system.

• Journal of Life Science
• /
• v.28 no.12
• /
• pp.1416-1423
• /
• 2018

With strong biotin binding affinity ($K_D=10^{-14}M$), the tetrameric feature of streptavidin could be used to increase the antigen binding activity of a camel heavy chain (VHH) antibody through their fusion, here stained with biotinylated horseradish peroxidase and subsequent immunoassays ELISA and Western blot analysis. For this application, we cloned the streptavidin gene amplified from the Streptomyces avidinii chromosome by PCR, and this was fused to the gene of the 8B9 VHH antibody which is specific to green fluorescent protein (GFP) antigens. To express a soluble fusion protein in Escherichia coli, we used the pUC119 plasmid-based expression system which uses the lacZ promoter for induction by IPTG, the pelB leader sequence at the N-terminus for secretion into the periplasmic space, and six polyhistidine tags at the C-terminus for purification of the expressed proteins using an $Ni^+$-NTA-agarose column. Although streptavidin is toxic to E. coli because of its strong biotin binding property, this soluble fusion protein was expressed successfully. In SDS-PAGE, the size of the purified fusion protein was 122.4 kDa in its native condition and 30.6 kDa once denatured by boiling, suggesting the tetramerization of the monomeric subunit by non-covalent association through the streptavidin moiety fusing to the 8B9 VHH antibody. In addition, this fusion protein showed biotin binding activity similar to streptavidin as well as GFP antigen binding activity through both ELISA and Western blot analysis. In conclusion, the protein resulting from the fusion of an 8B9 VHH antibody with streptavidin was successfully expressed and purified as a soluble tetramer in E. coli; it showed both biotin and GFP antigen binding activity suggesting the possible production of a tetrameric and bifunctional VHH antibody.

• KSBB Journal
• /
• v.14 no.1
• /
• pp.82-90
• /
• 1999

In order to eventually fabricate an analytical system for infectious microorganisms, we synthesized major immunochemical components, utilized them for the construction of model system, and investigated an assay concept for bacterial whole cells. For the preparation of system components, a polyclonal antibody, against Salmonella thompson as model analyte, purified by immuno-affinity chromatography was used to chemically link to streptavidin or an enzyme, horseradish peroxidase(HRP). The antibody and streptavidin was modified with sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate and N-succinimidyl-3-[2-pyridyldithio]propionate(subsequently activated by dithiotheritol), respectively. The modified components were reacted to synthesize antibody-streptavidin conjugates which were then purified on a two-layer chromatography column of diaminobiotin gel and Sephadex G-100. For antibody-HRP conjugates, HRP molecules were activated by $NalO_4$ oxidation and then coupled to immunoglobulin. After stabilizing with ($NaCNBH_3$, the conjugates were purified by size exclusion chromatography on Biogel A5M column. To devise a model system, such produced components were combined with a dot-blotter in which a nitrocellulose membrane($12{\mu}m$ pre size) with immobilized biotin was already located. The analyte (S. thompson cells) was reacted with the both antibody conjugates in a liquid phase, and the complexes formed were captured on the membrane surfaces by applying vacuum in the bottom compartment of the blotter to invoke biotin-streptavidin reaction. Under optimal conditions, the system enabled to identify the analytical concept for bacterial whole cells, and the lower limit of detection was approximately $1{\mu}g/m{\ell}$($10^5-10^6$ cells/m$m{\ell}$). The controlling factors were the concentrations of each antibody conjugate that caused agglutination in the presence of analyte as they increased.

• Korean Journal of Computational Design and Engineering
• /
• v.12 no.4
• /
• pp.298-307
• /
• 2007

In recent years, researchers are actively investigating new methods that are applicable for manufacturing micrometer to nanometer scale structures. Among them, optical tweezers that can manipulate microscopic objects using a laser is receiving one of the key attentions. Optical tweezers have been used actively in the field of science. For example, for measuring mechanical characteristics in the scale of piconewtons or for manipulating and sorting large numbers of particles, bacteria, cells. etc. However, little works have been reported for "manufacturing" objects. In this paper, we present a new method for manufacturing micrometer scale structures using micrometer scale biotin coated polystyrene particles. Particles will be controlled with a user interface that utilizes a CyberGlove and glued together by the bonding force between biotin and streptavidin.

• Biotechnology and Bioprocess Engineering:BBE
• /
• v.9 no.2
• /
• pp.112-117
• /
• 2004

The performance of an immuno-analytical system can be assessed in terms of its analytical sensitivity, i.e., the detection limit of an analyte, which is determined by the amount of analyte molecules bound to the capture antibody that has been immobilized onto a solid surface. To increase the number of the binding complexes, we have investigated a site-directed immobilization of an antibody that has the ability to resolve a current problem associated with a random arrangement of the insolubilized immunoglobulin. The binding molecules were chemically reduced to produce thiol groups that were limited at the hinge region, and then, the reduced products were coupled to biotin. This biotinylated antibody was bound to a streptavidin-coated surface via the streptavidin-biotin reaction. This method can control the orientation of the antibody molecules present on a solid surface and also can significantly reduce the possibility of steric hindrance in the antigen-antibody reactions. In a two-site immunoassay, the introduction of the site-directly immobilized antibody as the capture enhanced the sensitivity of analyte detection approximately 10 times compared to that of the antibody randomly coupled to biotin. Such a novel approach would offer a protocol of antibody immobilization in order for the possibility of constructing a high performance immunochip.

• Korean Journal of Microbiology
• /
• v.26 no.3
• /
• pp.149-154
• /
• 1988

Japanese Encephalitis Virus(JEV) can be detected conveniently by the use of biotinylated cDNA probe. To prepare biotinylated probe aminoallyl dUTP was first synthesized chemically to reverse transcribe the virial RNA. The allylamine-labeled cDNA was then converted to the biotin-cDNA by the reaction with an activated biotin ester, NHS-ACA-biotin. The JEV genomic RNA was hybridized to the biotinylated cDNA probe on nitrocellulose filter and visualized colorimetrically by streptavidin complexes with alkaline phosphatase polymer. Sensitivity of the detection system was determined by estimating the amount of the JEV genomic RNA through comparison with signals generated from the biotinylated and $^{32/P}$ -labeled probes. It was found that the biotin probe was as sensitive as $^{32/P}$ -cDNA probe which can detect 50pgs of the target RNA.

• Biotechnology and Bioprocess Engineering:BBE
• /
• v.10 no.6
• /
• pp.505-509
• /
• 2005

For the purpose of developing a direct label-free electrochemical detection system, we have systematically investigated the electrochemical signatures of each step in the preparation procedure, from a bare gold electrode to the hybridization of label-free complementary DNA, for the streptavidin-modified electrode. For the purpose of this investigation, we obtained the following pertinent data; cyclic voltammogram measurements, electrochemical impedance spectra and square wave voltammogram measurements, in $Fe(CN)_6^{3-}/Fe(CN)_6^{4-}$ solution (which was utilized as the electron transfer redox mediator). The oligonucleotide molecules on the streptavidin-modified electrodes exhibited intrinsic redox activity in the ferrocyanide-mediated electrochemical measurements. Furthermore, the investigation of electrochemical electron transfer, according to the sequence of oligonucleotide molecules, was also undertaken. This work demonstrates that direct label-free oligonucleotide electrical recognition, based on biofunctional streptavidin-modified gold electrodes, could lead to the development of a new biosensor protocol for the expansion of rapid, cost-effective detection systems.