• 한국생물공학회:학술대회논문집
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• 2003.10a
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• pp.548-548
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• 2003

• 한국생물공학회:학술대회논문집
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• 2005.04a
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• pp.375-375
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• 2005

• Journal of Microbiology and Biotechnology
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• v.11 no.4
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• pp.543-551
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• 2001

The vascular endothelial growth factor (VEGF), or VEGF-A, is intimately involved in both physiological and pathological forms of angiogenesis. VEGF-A is now recognized as the founding member of a family of growth factors that has expanded to include VEGF-B, VEGF-C, VEGF-D, VEGF-E, and placental growth factor (PIGF). This family of cytokines binds differentially to at least three receptor tyrosine kinases, however, the extent to which family members other than VEGF-A contribute to physiological and pathological angiogenesis remains unclear. Issues that are of relevance include uncertainty regarding the consequences of signaling through VEGF - RI in particular, and the ability of some family members to heterodimerize, leading to the possibility ofheterodimeric receptor complexes. Structural characterization is one approach that can be used to address these issues, however, the vast majority of previous structure-function studies have only focused on VEGF-A. While these studies may provide some clues regarding the structural basis of the interaction of other family members with their receptors, studies using the ligands themselves are clearly required if highly specific interactions are to be revealed. With the recent progress toward refolding and purifying substantial' quantities of other VEGF family members, such structural studies are now possible. Here, these ~ssues are addressed with a particular emphasis on VEGF-B and its receptors.

• BMB Reports
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• v.32 no.6
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• pp.521-528
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• 1999

The folding of recombinant hepatitis B virus X-protein (rHBx) solubilized from Escherichia coli inclusion bodies was investigated. By sequential dialysis of urea, rHBx was folded into its native structure, which was demonstrated by the efficacy of its transcriptional activation of the adenovirus major late promoter (MLP), fluorescence spectroscopy, and circular dichroism (CD) analysis. The decrease in CD values at 220 nm and a corresponding blue shift of the intrinsic fluorescence emission confirmed the ability of rHBx to refold in lower concentrations of urea, yielding the active protein. Equilibrium and kinetic studies of the refolding of rHBx were carried out by tryptophan fluorescence measurements. From the biphasic nature of the fluorescence curves, the existence of stable intermediate states in the renaturation process was inferred. Reverse phase-high performance liquid chromatography (RP-HPLC) analysis further demonstrated the existence of these intermediates and their apparent compactness.

• BMB Reports
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• v.34 no.1
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• pp.21-27
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• 2001

The structural stability of brain pyrydoxine-5'-phosphate (PNP) oxidase and the catalytic properties of the monomeric species were investigated. The unfolding of brain pyridoxine-5'-phosphate (PNP) oxidase by guanidine hydrochloride (GuHCl) was monitored by means of fluorescence and circular dichroism spectroscopy Reversible dissociation of the dimeric enzyme into subunits was attained by the addition of 2 M GuHCl. The perturbation of the secondary structure under the denaturation condition resulted in the release of the cofactor FMN. Separation of the processes of refolding and reassociation of the monomeric species was achieved by the immobilization method. Dimeric PNP oxidase was immobilized by the covalent attachment to Affi-gel 15 without any significant lass of its catalytic activity. Matrix-bound monomeric species were obtained from the reversible refolding processes. The matrix bound-monomer was found to be catalytically active, possessing only a slightly decreased specific activity when compared to the refolded dimeric enzyme. In addition, limited chymotrypsin digestion of the oxidase yields two fragments of 12 and 161 kDa with a concomitant increase of catalytic activity The catalytically active fragment was isolated by ion exchange chromatography and analyzed for association of two subunits using the FPLC gel filtration analysis. The retention time indicated that the catalytic fragment of 16 kDa behaves as a compact monomer. Taken together, these results are consistent with the hypothesis that the native quaternary structure of PNP oxidase is not a prerequisite for catalytic function, but it could play a role in the regulation.

• Microbiology and Biotechnology Letters
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• v.41 no.1
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• pp.1-7
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• 2013

Ionic liquids (ILs) have been widely recognized as environmentally benign solvents. Their unique properties, including negligible vapor pressure, non-flammability, a wide liquid range and their tunable physicochemical properties by proper selection of cations and anions, make them attractive green solvents in a variety of fields such as organic synthesis/catalysis, extraction/ separation, and electrochemistry, amongst others. In this paper, the recent technological developments and their prospects in the application of ILs in microbiology and biochemical engineering, including enzymatic reactions, protein folding/refolding and biomass dissolution, are discussed.

• Korean Journal of Food Science and Technology
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• v.25 no.3
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• pp.277-281
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• 1993

To elucidate the conformational stability of proteins in colloidal food system, molecular properties of various proteins such as chemically modified ${\beta}-lactoglobulin$, bovine serum albumin (BSA) structural intermediates, and ${\beta}-casein$ under chaotropic conditions, were examined using circular dichroism, SS bond content, and hydrodynamic radius determination. As refolding time increases, BSA intermediates approach the conformation of native BSA. And succinylation made ${\beta}-lactoglobulin$ have more aperiodic structure by increasing net negative charge. Also, under chaotropic conditions, the conformation of P-casein was affected by hydrophobic interactions. This study clearly indicates that hydrophobic interactions and electrostatic interactions are major contributing factors in conformational stability of proteins.

• Bulletin of the Korean Chemical Society
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• v.30 no.5
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• pp.1101-1106
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• 2009

Multivalent and multi-specific antibodies can provide valuable tools for bio-medical research, diagnosis and therapy. In antigen-antibody interactions, the avidity of antibodies depends on the affinity and the number of binding sites.$^1$ As artificial multivalent antibody agents, single chain Fv-streptavidin fusion tetramer proteins $(scFv-SA)_4$ have been previously tested.$^{1,\;2}$ Although, the Fab domain is known to be more stable than scFv in animal models,$^{3,\;4}$ it has never been used to make a multivalent agent with a streptavidin fusion. In this study, we prepared tetra-valent $(Fab-cSA)_4$ by fusing Fab with core streptavidin (cSA). This molecule was made using inclusion body production, refolding and chromatography purification. Affinities of the Fab-cSA tetramer and a scFv-cSA tetramer to a cell surface antigen were compared by ELISA using biotin-HRP. The Fab-cSA tetramer showed higher binding avidity than the scFv-cSA tetramer. The higher binding avidity of the Fab-cSA tetramer demonstrates its potential as a therapeutic agent for target-specific antibody therapy.

• BMB Reports
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• v.28 no.6
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• pp.477-483
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• 1995

The recombinant human interferon alpha 2a ($rhIFN-{\alpha}2a$), expressed in Saccharomyces cerevtsiae, was purified from insoluble aggregates. The inclusion body of $rhIFN-{\alpha}$ was solubilized by guanidine salt in the presence of disulfide reducing agent. The refolding of denatured $rhIFN-{\alpha}2a$ was achieved by simple dilution. The authentic interferon alpha, which has two correctly matched disulfide bonds, was seperated from incompletely oxidized $IFN-{\alpha}$ and dimeric $IFN-{\alpha}$ by use of a CM-Sepharose column, followed by size exclusion columns at two different pH conditions. The purified protein has been subjected to detailed physicochemical characterization including sequence determination. Unlike other $rhIFN-{\alpha}2a$ from E. coli reported, the $rhIFN-{\alpha}2a$ from S. cerevisiae has no methionine residue at its N-terminus originating from the start codon, ATG. The pI of the protein was determined to be 6.05 with a single band in the pI gel, which demonstrated that the purified $rhIFN-{\alpha}$ was homogeneous. The structural study using circular dichroism showed that the protein retains its three dimensional structure in the wide range of pH conditions between pH 3 and 9, and only minor strucural deformation was observed at pH 1.0.

• Journal of Microbiology and Biotechnology
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• v.16 no.7
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• pp.1097-1103
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• 2006

The divalent antibody-toxins are expected to have increased binding avidities to target cells because of the two cell-binding domains. However, previous studies showed that the refolding yield of divalent antibody-toxin is very low, and it is assumed that homodimer formation of antibody-toxin is strongly interfered by the repulsion between the two large toxin domains that come close to each other during dimer formation. In this study, B3 antibody was used as a model antibody, and its Fab domain was used to construct three different kinds of Fab divalent molecules, $[B3(Fab)-toxin]_{2}$. The monomer Fab-toxin molecules were made by fusing the Fab domain of monoclonal antibody B3 to PE38, a truncated mutant form of Pseudomonas exotoxin (PE), and a connecting sequence that contained spacer amino acid sequence (G4S)n (n=l, 2, 3) was inserted between Fab and PE38. The prepared divalent molecules were $[Fab-S\;1,\;2,\;3-PE38]_{2}\;(=[Fab-SKPCIST-KAS(G_{4}S)nGGPE-PE38]_{2}\;(n=1,\;2,\;3))$, and they are derivatives of previously studied $[Fab-H2cys-PE38]_{2}\;(=[Fab-SKPCIST-KASGGPE-PE38]_{2})$. In $[Fab-Sl,\;2,\;3-PE38]_{2}$, two Fab-S1, 2, 3-PE38 monomers were covalently linked by the disulfide bond bridge made from cysteine in the -SKPCIST- sequence. The insertion of spacer amino acids after the disulfide bridge resulted in a 12-18 fold higher yield of dimer formation than previously constructed $[Fab-Hlcys-PZ38]_{2}[7]$, 3-4-fold higher than $[Fab-ext-PZ38]_{2}[25]$. These two molecules have less amino acid spacer sequence between the disulfide bridge and PE38 domain. The design of $[Fab-PE38]_{2}$ in this study gave molecules with a higher refolding yield. The results of cytotoxicity assay showed a higher cytotoxic effect of these divalent molecules than that of the monovalent scFv-PE38 molecule.