• 한국생물공학회:학술대회논문집
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• 2000.04a
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• pp.541-545
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• 2000

For the sustained release formulation of recombinant human growth hormone (rhGH), dissociable rhGH aggregates were microencapsulated within poly(D,L-lactic-co-glycolic acid) [PLGA] microparticles. rhGH aggregates with 2 - 3 m Particle diameter were first produced by adding a small volume of aqueous rhGH solution into a partially water miscible organic solvent phase(ethyl acetate) containing PLGA. These rhGH aggregates were then microencapsulated within PLGA polymer phase by extracting ethyl acetate into an aqueous phase pre-saturated with ethyl acetate. The resultant microparticles were 2 - 3 m in diameter similar to the size of rhGH aggregates, suggesting that PLGA polymer was coated around the protein aggregates. Release profiles of rhGH from these microparticles were greatly affected by changing the volume of the incubation medium. The release rhGH species consisted of mostly monomeric form with having a correct conformation. This study reveals that sustained rhGH release could be achieved by microencapsulating reversibly dissociable protein aggregates within biodegradable polymers.

• Journal of Microbiology and Biotechnology
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• v.14 no.4
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• pp.783-788
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• 2004

Marker proteins of genetically-modified organisms (GMO) and their antibodies were prepared and characterized as major components of an analytical system. We selected two GMO markers, neomycin phosphotransferase II and 5- enolpyruvylshikimate-3-phosphate synthase, and produced them from E. coli employing genetic recombination technology. After purification, their structural conformation and binding affinities to the respective antibodies were characterized. The results showed that the recombinant proteins were identical with commercially obtained reference proteins. We further used them as immunogens to raise polyclonal antibodies capable of discriminating GMO containing protein from non-GMO. Well-characterized marker proteins and antibodies will be valuable as immunoreagents in constructing analytical systems such as biosensors and biochips to measure quantities of GMO.

• Journal of the Korean Magnetic Resonance Society
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• v.8 no.1
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• pp.19-27
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• 2004

C-terminal fragments of APP (APP-CTs), that contain complete Abeta sequence, are found in neuritic plaques, neurofibrillary tangles and the cytosol of lymphoblastoid cells obtained from AD patients. CT16, Lys649-Asp664 (KKQYTSIHHGVVEVD) has been known as the most toxic part in the C-terminal fragment of amyloid precursor protein (APP). The solution structure of CT16 was investigated using NMR spectroscopy in various membrane-mimicking environments. According to Circular Dichroim (CD) spectra, CT16 has a random structure in aqueous solution, while conformational change was induced by addition of TFE and SDS micelle. Tertiary structure as determined by NMR spectroscopy shows that CT16 has a ${\beta}$-turn conformation in trifluoroethanol-containing aqueous solution.

• Interdisciplinary Bio Central
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• v.1 no.1
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• pp.3.1-3.6
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• 2009

Introduction: GTPases known as translation factor play a vital role as ribosomal subunit assembly chaperone. The bacterial Obg proteins ($Spo{\underline{0B}}$-associated ${\underline{G}}TP$-binding protein) belong to the subfamily of P-loop GTPase proteins and now it is considered as one of the new target for antibacterial drug. The majority of bacterial Obgs have been commonly found to be associated with ribosome, implying that these proteins may play a fundamental role in ribosome assembly or maturation. In addition, one of the experimental evidences suggested that Bacillus subtilis Obg (BsObg) protein binds to the L13 ribosomal protein (BsL13) which is known to be one of the early assembly proteins of the 50S ribosomal subunit in Escherichia coli. In order to investigate binding mode between the BsObg and the BsL13, protein-protein docking simulation was carried out after generating 3D structure of the BsL13 structure using homology modeling method. Materials and Methods: Homology model structure of BsL13 was generated using the EcL13 crystal structure as a template. Protein-protein docking of BsObg protein with ribosomal protein BsL13 was performed by DOT, a macro-molecular docking software, in order to predict a reasonable binding mode. The solvated energy minimization calculation of the docked conformation was carried out to refine the structure. Results and Discussion: The possible binding conformation of BsL13 along with activated Obg fold in BsObg was predicted by computational docking study. The final structure is obtained from the solvated energy minimization. From the analysis, three important H-bond interactions between the Obg fold and the L13 were detected: Obg:Tyr27-L13:Glu32, Obg:Asn76-L13:Glu139, and Obg:Ala136-L13:Glu142. The interaction between the BsObg and BsL13 structures were also analyzed by electrostatic potential calculations to examine the interface surfaces. From the results, the key residues for hydrogen bonding and hydrophobic interaction between the two proteins were predicted. Conclusion and Prospects: In this study, we have focused on the binding mode of the BsObg protein with the ribosomal BsL13 protein. The interaction between the activated Obg and target protein was investigated with protein-protein docking calculations. The binding pattern can be further used as a base for structure-based drug design to find a novel antibacterial drug.

• BMB Reports
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• v.43 no.8
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• pp.561-566
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• 2010

Though protein transduction domains (PTDs) are well known for the delivery of exogenous therapeutic proteins into living cells, the overall low efficiency of transduction is a serious obstacle. We investigated the effect of bog blueberry anthocyanins (BBA) on protein transduction efficiency and found that BBA enhanced the transduction efficiencies of Tat-SOD fusion protein into HeLa cells and mice skin. The enzymatic activities in the cells and skin tissue in the presence of BBA were markedly increased compared to controls. Further, BBA did not demonstrate any cell toxicity at various concentrations. Although the mechanism is not fully understood, we suggest that BBA might alter the conformation of the membrane, which would indicate that BBA can be used as a protein transduction enhancer for the efficient delivery of therapeutic proteins for a variety of disorders.

• Journal of Life Science
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• v.28 no.10
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• pp.1140-1146
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• 2018

Dual-specificity phosphatases (DUSPs) play a role in cell growth and differentiation by modulating mitogen-activated protein kinases. DUSPs are considered targets for drugs against cancers, diabetes, immune diseases, and neuronal diseases. Part of the DUSP family, DUSP19 modulates c-Jun N-terminal kinase activity and is involved in osteoarthritis pathogenesis. Here, we report screening of cavity-creating mutants and the crystal structure of a cavity-creating L75A mutant of DUSP19 which has significantly enhanced enzyme activity in comparison to the wild-type protein. The crystal structure reveals a well-formed cavity due to the absent Leu75 side chain and a rotation of the active site-bound sulfate ion. Despite the cavity creation, residues surrounding the cavity did not rearrange significantly. Instead, a tightened hydrophobic interaction by a remote tryptophan residue was observed, indicating that the protein folding of the L75A mutant is stabilized by global folding energy minimization, not by local rearrangements in the cavity region. Conformation of the rotated active site sulfate ion resembles that of the phosphor-tyrosine substrate, indicating that cavity creation induces an optimal active site conformation. The activity enhancement by an internal cavity and its structural information provide insight on allosteric modulation of DUSP19 activity and development of therapeutics.

• BMB Reports
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• v.39 no.3
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• pp.319-328
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• 2006

SecA protein, a cytoplasmic ATPase, plays a central role in the secretion of signal peptide-containing proteins. Here, we examined effects of signal peptide and ATP on the oligomerization, conformational change, and membrane binding of SecA. The wild-type (WT) signal peptide from the ribose-binding protein inhibited ATP binding to soluble SecA and stimulated release of ATP already bound to the protein. The signal peptide enhanced the oligomerization of soluble SecA, while ATP induced dissociation of SecA oligomer. Analysis of SecA unfolding with urea or heat revealed that the WT signal peptide induces an open conformation of soluble SecA, while ATP increased the compactness of SecA. We further obtained evidences that the signal peptide-induced oligomerization and the formation of open structure enhance the membrane binding of SecA, whereas ATP inhibits the interaction of soluble SecA with membranes. On the other hand, the complex of membrane-bound SecA and signal peptide was shown to resume nucleotide-binding activity. From these results, we propose that the translocation components affect the degree of oligomerization of soluble SecA, thereby modulating the membrane binding of SecA in early translocation pathway. A possible sequential interaction of SecA with signal peptide, ATP, and cytoplasmic membrane is discussed.

• Journal of Life Science
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• v.13 no.5
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• pp.751-755
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• 2003

Cyclic AMP receptor proteins(CRP) activate many genes in Escherichia coli by binding of cAMP with not fully known mechanism. CRP existed as apo-CRP in the absence of cAMP, $CRP;(cAMP)_2$$_2$ at low(micromolar) cAMP concentration, or $CRP;(cAMP)_4$ at high(millimolar) concentration of cAMP. This study is designed to measure the thermal stability of S83G CRP, which substituted glycine for serine at amino acid 83 position, with CD spectrapolarimeter at 222nm by the constant elevation of temperature from $20^{\circ]C\; to\; 90^{\circ}C\; at\; 1^{\circ}C/min$. The non-linear regression analysis showed that melting temperatures were 68.4, 72.0, and $82.3^{\circ}C$ for no cAMP, 0.1mM cAMP, and 5mM cAMP, respectively. Result showed the strong thermal stability of CRP by binding of additional cAMP molecules to region between the hinge region and helix-turn-helix(HTH) motif at 5mM cAMP concentration.

• Proceedings of the Korean Institute of Electrical and Electronic Material Engineers Conference
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• 2001.11b
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• pp.3-6
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• 2001

In common globular proteins, the native form is in its most stable state. However, the native form of inhibitory serpins (serine protease inhibitors) and some viral membrane fusion proteins is in a metastable state. Metastability in these proteins is critical to their biological functions. Our previous studies revealed that unusual interactions, such as side-chain overpacking, buried polar groups, surface hydrophobic pockets, and internal cavities are the structural basis of the native metastability. To understand the mechanism by which these structural defects regulate protein functions, cavity-filling mutations of a 1-antitrypsin, a prototype serpin, were characterized. Increasing conformational stability is correlated with decreasing inhibitory activity. Moreover, the activity loss appears to correlate with the decrease in the rate of the conformational switch during complex formation with a target protease. We also increased the stability of a 1-antitrypsin greatly via combining various stabilizing single amino acid substitutions that were distributed throughout the molecule. The results showed that a substantial increase of stability, over 13 kcal/mol, affected the inhibitory activity with a correlation of 11% activity loss per kcal/mol. The results strongly suggest that the native metastability of proteins is indeed a structural design that regulates protein functions and that the native strain of a 1-antitrypsin distributed throughout the molecule regulates the inhibitory function in a concerted manner.

• Journal of Microbiology and Biotechnology
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• v.9 no.3
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• pp.276-281
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• 1999

In order to determine the functional region of the antifungal protein (AFP) isolated from Aspergillus giganteus responsible for growth inhibitory activity and the promotion of phospholipid vesicle aggregation, overlapping peptides covering the complete sequence of AFP were synthesized. The antibiotic activity against bacterial, fungal, and tumor cells, and the vesicle-aggregation activity of the synthetic peptides were investigated. The AFP functional sequence responsible for antibiotic and vesicle-aggregation activity was determined to be located within the region between AFP residues 19 to 32. AFP (19-32) exhibited an a-helical conformation in a cell membrane-like environment. AFP (19-32) displayed potent antibiotic activity against bacterial, fungal, and tumor cells without peptide toxicity as indicated by hemolysis. Accordingly, AFP (19-32) could be used as a good model for the design of effective antibiotic agents with powerful antibiotic activity yet without any cytotoxic effects against the host organism.