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CaCl<sub>2</sub>/EDTA 및 비이온성 계면활성제 활용 Inclusion Body 정제법을 이용한 BA-RGD 단백질의 생산
송우호,변창우,윤민호,엄지훈,최유성,Song, Wooho,Byun, Chang Woo,Yoon, Minho,Eom, Ji Hoon,Choi, Yoo Seong 한국생물공학회 2015 KSBB Journal Vol.30 No.6
The limited productivity of natural shell matrix proteins has hampered the investigation of their biochemical properties and practical applications, although biominerals in nature obtained by organic-inorganic assemblies have attractive mechanical and biological properties. Here, we prepared a vector for the expression of a fusion protein of a shell matrix protein from Pinctada fucata (named as GRP_BA) with the GRGDSP residue. The fusion protein of BA-RGD was simply produced in E. coli and purified through sequential steps including the treatment with $CaCl_2$ and EDTA solution for cell membrane washing, mechanical cell disruption and the application of non-ionic surfactant of Triton X-100 for BA-RGD inclusion body washing. The production yield was approximately 60 mg/L, any other protein band was not observed in SDS-PAGE and it was estimated that above 97% endotoxin was removed compared to the endotoxin level of whole cell. This study showed this simple and easy purification approach could be applied to the purification of BA-RGD fusion protein. It is expected that the protein could be utilized for the preparation of biominerals in practical aspects.
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CaCl₂/EDTA 및 비이온성 계면활성제 활용 Inclusion Body 정제법을 이용한 BA-RGD 단백질의 생산
송우호(Wooho Song),변창우(Chang Woo Byun),윤민호(Minho Yoon),엄지훈(Ji Hoon Eom),최유성(Yoo Seong Choi) 한국생물공학회 2015 KSBB Journal Vol.30 No.6
The limited productivity of natural shell matrix proteins has hampered the investigation of their biochemical properties and practical applications, although biominerals in nature obtained by organic-inorganic assemblies have attractive mechanical and biological properties. Here, we prepared a vector for the expression of a fusion protein of a shell matrix protein from Pinctada fucata (named as GRP_BA) with the GRGDSP residue. The fusion protein of BA-RGD was simply produced in E. coli and purified through sequential steps including the treatment with CaCl₂ and EDTA solution for cell membrane washing, mechanical cell disruption and the application of non-ionic surfactant of Triton X-100 for BA-RGD inclusion body washing. The production yield was approximately 60 mg/L, any other protein band was not observed in SDS-PAGE and it was estimated that above 97% endotoxin was removed compared to the endotoxin level of whole cell. This study showed this simple and easy purification approach could be applied to the purification of BA-RGD fusion protein. It is expected that the protein could be utilized for the preparation of biominerals in practical aspects.
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재조합단백질 GRP_BA 및 GG1234를 이용한, 상온상압조건에서의 In vitro 탄산칼슘 결정화
손채연,송우호,최현석,최유성 한국화학공학회 2019 Korean Chemical Engineering Research(HWAHAK KONGHA Vol.57 No.2
바이오미네랄의 독특한 구조 및 생물학적 물성은 다양한 의료 및 산업용 분야에서 활용할 수 있는 뛰어난 잠재력을 지니고 있어 최근 관심이 증대되고 있다. 껍질 메트릭스 단백질에 의해 조절되는 탄산칼슘 생광물화는 이러한 바이오 미네랄의 생성 메커니즘을 이해하기 위한 대표적인 모델로 활용되고 있다. 본 연구에서는 진주조개 프리즘층에 존재하는 껍질 메트릭스 단백질인 GRP_BA 재조합단백질과 껍질 메트릭스 단백질의 특성과 유사한 인공단백질 GG1234를 이용하여, 상온상압 조건에서 in vitro 탄산칼슘 결정화를 진행하였다. 대표적인 탄산칼슘 결정화 방법인 CaCl2 용액과 (NH4)2CO3 증기를 활용하였을 때, 두 단백질 모두 상온상압 조건에서 전형적인 능면체의 방해석 결정 성장을 저해하였고, 하위단위의 작은 방해석 결정이 뭉쳐진 장미모양리본 형태의 구형 방해석 성장을 유도하였다. 이러한 실험결과는 두 단백질에서 나타나는 블록으로 구성된 무정형 단백질의 특성에 의해 야기된 것으로 추정되며, 이러한 측면에서 본 연구는 껍질 메트릭스 단백질에 의해 조절되는 탄산칼슘 생광물화 현상의 이해를 높이는 데 기여할 것으로 판단된다. The exquisite structure and attractive biological properties of biominerals have great potential and increased interest for use in a wide range of medical and industrial applications. Calcium carbonate biomineralization, mainly controlled by shell matrix proteins, has been used as a representative model to understand the biomineralization mechanism. In this study, in vitro calcium carbonate crystallization was carried out under room temperature and atmospheric pressure using recombinant shell matrix protein GRP_BA and artificial shell matrix protein GG1234. Both proteins inhibited the growth of typical rhombohedral calcite crystals in the calcium carbonate crystallization using CaCl2 solution and (NH4)2CO3 vapor, and spherulitic calcite crystals with rosette-like structures were synthesized in both the presence of GRP_BA and GG1234. These results might be caused by the properties of block-like domain structure and intrinsically disordered proteins. We expect that this study can contribute to enhance understanding of the calcium carbonate biomineralization controlled by shell matrix proteins.
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문은주,강은수,송우호,김범진,차형준,최유성 한국화학공학회 2023 Korean Journal of Chemical Engineering Vol.40 No.2
Chitosan-based nanofibers have become attractive biomaterials for wound healing and dressing applications based on their intrinsic biocompatibility, biodegradability, and antibacterial properties. However, the unstable spinnability of chitosan-based nanofibers has impeded further applications. In this paper, a fatty acid amide oleamide was used as a blending material for nanofiber fabrication. The addition of oleamide into chitosan moderately decreased the viscosity of the electrospinning solution, resulting in enhanced spinnability when constructing chitosan/oleamide blended nanofibers. Remarkably, the 1:0.5 ratio of chitosan/oleamide nanofibers exhibited relatively high hydrophobicity, decreased tensile strength, and increased elongation at break compared to chitosan-only nanofiber. The nanofiber showed similar and slightly higher cell adhesion in the in vitro cell culture with mouse preosteoblast MC3T3-E1 and fibroblast NIH/3T3 cells, respectively; however, the cell proliferation levels were decreased on the blended nanofiber surfaces, presumably due to their increased hydrophobicity. These results suggest that chitosan/oleamide nanofibers with high spinnability can be applied to the preparation of wound dressing membranes or patches with intrinsic antibacterial properties and moderate hydrophobicity. We expect that oleamide, which has lubricant and antibacterial properties, can be utilized as a blending component of chitosan-based nanofibers for biomaterial and tissue engineering applications.
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황소라(Sora Hwang),남진오(Jin-Oh Nam),이병진(Byung-Jin Lee),송우호(Woo-Ho Song),이창수(Chang-Soo Lee) 한국생물공학회 2012 KSBB Journal Vol.27 No.4
Emulsions are mixture of immiscible liquids in which one is dispersed in all over the other. They have been applied to many applications including cosmetics, foods, drug delivery system (DDS), fine chemicals, and chemical separations. Especially, emulsion technology is one of the most useful technique to formulate cosmetics such as eye cream, foundation, and foam cleansing. In general, the emulsions can be generated by mechanical agitation of two immiscible fluids. However, the emulsions obtained by conventional method have limited in stability, monodispersity, and complicate process. We describe here preparation techniques of representative cosmetic emulsions such as liposome, liquid crystal emulsion, nanoemulsion, multiple emulsion, and pickering emulsion. Furthermore, various factors which can control the physical properties of each cosmetic emulsions are briefly discussed.
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