• BMB Reports
• /
• 제49권11호
• /
• pp.585-586
• /
• 2016

Extracellular vesicles (EVs) are natural carriers of biomolecules that play central roles in cell-to-cell communications. Based on this, there have been various attempts to use EVs as therapeutic drug carriers. From chemical reagents to nucleic acids, various macromolecules were successfully loaded into EVs; however, loading of proteins with high molecular weight has been huddled with several problems. Purification of recombinant proteins is expensive and time consuming, and easily results in modification of proteins due to physical or chemical forces. Also, the loading efficiency of conventional methods is too low for most proteins. We have recently proposed a new method, the so-called exosomes for protein loading via optically reversible protein-protein interaction (EXPLORs), to overcome the limitations. Since EXPLORs are produced by actively loading of intracellular proteins into EVs using blue light without protein purification steps, we demonstrated that the EXPLOR technique significantly improves the loading and delivery efficiency of therapeutic proteins. In further in vitro and in vivo experiments, we demonstrate the potential of EXPLOR technology as a novel platform for biopharmaceuticals, by successful delivery of several functional proteins such as Cre recombinase, into the target cells.

• BMB Reports
• /
• 제29권5호
• /
• pp.455-461
• /
• 1996

A serine proteinase was purified from Acanthamoeba culbertsoni by 41~80% ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography and gel filtration chromatography. The molecular weight of the purified enzyme was estimated to be 108.0 kDa by gel filtration chromatography and 54.0 kDa by SDS-PAGE. Therefore, the purified enzyme seemed to be a dimer. Isoelectric point was 4.5. The enzyme activity was highly inhibited by the serine proteinase inhibitors diisopropyl fluorophosphate (OFP) and phenylmethyl sulfonylfluoride (PMSF). It had a narrow pH optimum of 6.5~7.5 with a maximum at pH 7.0. These data suggested that the purified enzyme was a neutral serine proteinase. Optimal temperature was $37^{\circ}C$. It was stable for at least 16 h at $4^{\circ}C$ and $37^{\circ}C$, but it was rapidly inactivated at $65^{\circ}C$ The activity of the purified enzyme was not influenced significantly by $Mg^{2+}$, $Mn^{2+}$, $Zn^{2+}$ or $Ca^{2+}$. However, the enzyme activity was highly inhibited by $Hg^{2+}$ The enzyme degraded type I collagen and fibronectin, but not BSA, hemoglobin, lysozyme, immunoglobulin A or immunoglobulin G.

• Asian-Australasian Journal of Animal Sciences
• /
• 제26권9호
• /
• pp.1347-1358
• /
• 2013

Membrane technology has revolutionized the dairy sector. Different types of membranes are used in the industry for various purposes like extending the shelf life of milk without exposure to heat treatment, standardization of the major components of milk for tailoring new products as well increasing yield and quality of the dairy products, and concentrating, fractionation and purification of milk components especially valuable milk proteins in their natural state. In the cheese industry, membranes increase the yield and quality of cheese and control the whey volume, by concentrating the cheese milk. With the advancement of newer technology in membrane processes, it is possible to recover growth factor from whey. With the introduction of superior quality membranes as well as newer technology, the major limitation of membranes, fouling or blockage has been overcome to a greater extent.

• 한국세라믹학회지
• /
• 제54권2호
• /
• pp.114-120
• /
• 2017

In underdeveloped countries, many people suffer from water shortage due to the absence of water supply service. Although water purifiers have provided support in such situations, it is not easy to maintain water filters without a continuous supply of consumable filters. To obtain a sustainable drinking water source, appropriate technology of water treatment is necessary. Herein, a low cost water purification system was developed using natural raw materials. A non-electric water treatment system was developed using filtration through an Onggi filter, which is a type of Korean traditional earthenware with a microporous surface. The porosity and flux of the prepared Onggi filter were 29.06% and 31.63 LMH, respectively. After purification of water with total dissolved solids of 10.4 mg/L and turbidity of 100 NTU, the total dissolved solids and turbidity of the water treated using the Onggi filter decreased by 12% and 99.8%, respectively.

• 한국환경농학회지
• /
• 제25권1호
• /
• pp.25-33
• /
• 2006

농어촌 등 소규모로 발생되는 생활하수를 자연친화적으로 처리하기 위한 대책의 일환으로 인공습지에 적용되는 최적의 수생식물을 선발하기 위하여 다년생 수생식물 10종을 하수처리장에 식재한 후 하수 처리효율, 처리시기별 수생식물의 생육상 및 무기성분 흡수량 등을 조사하였다. 인공습지 하수처리장에서 하수중 BOD, COD, T-N 및 T-P의 수처리효율은 호기성조를 통과한 호기성조 처리수에서 각각 92%, 74%, 25% 및 57%이었고, 혐기성조를 통과한 방류수에서 각각 96%, 84%, 44% 및 71%이었다. 수생식물 생육 150일 후의 질소 및 인 흡수량은 호기성조에서는 물억새가 각각 17.7 및 2.41 g/plant로 가장 높았으며, 혐기성조에서는 큰고랭이가 각각 8.7및 1.1 g/plant로 가장 높았다. 인공습지 하수처리장의 호기성조에서 최적 수생식물은 물억새>달뿌리풀>갈대의 순이었고, 이들 수생식물은 대부분 뿌리의 발육이 좋은 심근성 수생식물이었고, 여재층 내로 산소를 공급하기 쉬운 통기조직이 발달된 수생식물들이었다. 인공습지 하수처리장의 혐기성조에서 최적 수생식물은 줄>큰고랭이>부들>노랑꽃창포>삿갓사초의 순이었고, 이들 수생식물은 대부분 뿌리의 발육보다 줄기와 잎의 발육이 좋은 천근성 수생식물로서 호기 성조의 최적 수생식물의 역할과는 달리 영양물질의 흡수량이 우수한 수생식물이 선정되었다.

• 한국환경농학회지
• /
• 제25권2호
• /
• pp.129-137
• /
• 2006

농촌 마을단위 하수처리장에서 오염물질의 존재형태별 처리 경향을 파악하여 하수처리장의 설계 및 시공시 부지면적 감소와 오염물질의 처리효율 극대화를 위한 기초자료로 활용하기 위하여 농촌 마을 단위 하수처리장을 호기성조와 혐기성조로 구분하여 시공한 다음 하수처리 경과시기별, 오염물질 부하량별 및 계절별로 각 오염물질의 존재형태별 수처리 효율을 조사하였다. 하수처리 경과시기에 따른 오염물질 처리효율을 조사한 결과 호기성조 처리수에서 하수처리 시간이 경과함에 따라 BOD, COD, TOC 및 SS처리량은 큰 폭으로 증가하였다. 처리된 오염물질의 존재형태를 조사한 결과 호기성조 및 혐기성조에서 BOD는 대부분 IBOD로, COD는 대부분 ICOD로, TOC는 대부분 STOC로, SS는 대부분 VSS로, T-N은 대부분 DTN 및 T-P는 대부분 DTP로 처리되었다. 부하량별 오염물질 처리효율을 조사한 결과 BOD 처리량은 호기성조와 방류수 모두에서 처리된 대부분의 BOD는 IBOD이었다. COD 처리량도 BOD와 유사하게 호기성조에서는 대부분 ICOD형태로 처리되었으나, 혐기성조에서는 ICOD와 SCOD가 비슷한 처리량을 보였다. 그러나 하수처리장에서 처리되는 TOC의 존재형태별 처리량은 BOD 및 COD와는 달리 호기성조 처리수와 방류수 모두에서 STOC의 처리량이 약간 많았다. 부하량별 SS 처리효율은 호기성조 처리수에서 95%정도 처리되어 대부분의 SS는 호기성조에서 처리되었으며, 처리되는 SS의 용존형태는 호기성조 처리수에서는 대부분 VSS로 처리되었고, 방류수에서는 VSS와 FSS가 비슷한 양으로 처리되었다. 부하량별 T-N 처리효율은 호기성조 처리수에서 32%정도이었고, 방류수에서 24%정도이었다. 또한 처리되는 T-N의 용존형태는 호기성조 처리수와 방류수 모두에서 DTN이 STN에 비해 월등히 많은 양이 처리되었다. 부하량별 T-P 처리효율은 호기성조에서 62%정도이었고, 혐기성조에서 14%정도로 대부분 호기성조에서 처리되었으며, 처리되는 T-P의 용존형태는 호기성조와 방류수 모두에서 대부분 DTP형태이었다. 계절별 오염물질 처리량을 조사한 결과 BOD, COD, TOC, SS, T-N 및 T-P 처리량은 여름과 가을이 봄과 겨울에 비해 처리량이 약간 증가되었으며, BOD, COD, TOC, SS, T-N 및 T-P는 4계절 모두 방류수의 처리효율이 각각 92, 89, 73, 95, 46 및 84%이상의 높은 처리효율을 나타내었다.

• 생명과학회지
• /
• 제29권4호
• /
• pp.492-498
• /
• 2019

기존 연구를 통하여 토양에서 분리한 Acinetobacter schindleri DYL129로부터 3개의 lipase 유전자(lipAD1, lipAD2와 lipAD3)들과 1개의 chaperone (lipBD) 유전자를 보고하였다. 본 연구에서는 각 유전자들의 발현을 위해서 pET32a(+)와 pGEX-6P-1 벡터에 클로닝하여 각각을 pETLAD1-3와 pETLBD 또는 pGEXLAD1-3와 pGEXLB로 명명하였으며, 단백질의 발현량은 pET 시스템을 사용할 때 1.5 배 정도 향상됨을 확인하였다. LipAD1과 LipAD2는 inclusion body 형태로 발현이 되었으며, LipAD3과 LipBD는 soluble type으로도 발현되었다. Inclusion body 형태의 LipAD1과 LipAD2는 고농도의 우레아를 처리하여 refolding 시켰다. LipAD1은 C4와 C2를, LipAD2는 C2와 C14를 그리고 lipAD3은 C2, C4와 C14를 기질로 잘 이용하는 것을 확인하였다. 그리고 모든 효소들은 $50^{\circ}C$에서 최적 활성을 나타내었다.

• Bulletin of the Korean Chemical Society
• /
• 제31권7호
• /
• pp.2019-2024
• /
• 2010

Insulin-like growth factor binding protein 5 (IGFBP-5) plays an important role in controlling cell survival, differentiation and apoptosis. Apoptosis can be induced by an extrinsic pathway involving the ligand-mediated activation of death receptors such as tumor necrosis factor receptor 1 (TNFR1). To determine whether IGFBP-5 and TNFR1 interact as members of the same apoptosis pathway, recombinant IGFBP-5 and TNFR1 were isolated. The expression and purification of the full-length TNFR1 and truncated IGFBP-5 proteins were successfully performed in E. coli. The binding of both IGFBP-5 and TNFR1 proteins was detected by surface plasmon resonance spectroscopy (BIAcore), fluorescence measurement, electron microscopy, and size-exclusion column (SEC) chromatography. IGFBP-5 indeed binds to TNFR1 with an apparent $K_D$ of 9 nM. After measuring the fluorescence emission spectra of purified IGFBP-5 and TNFR1, it was found that the tight interaction of these proteins is accompanied by significant conformational changes of one or both. These results indicate that IGFBP-5 acts potently as a novel ligand for TNFR1.

• BMB Reports
• /
• 제29권2호
• /
• pp.116-121
• /
• 1996

In earlier studies, the genes encoding Escherichia coli thioredoxin and Corynebacterium nephridii thioredoxin C-3 were fused via a common restriction site in the nucleotide sequence coding for the active site of the proteins to generate two chimeric thioredoxins, designated E-C3 (N to C-terminal) and C3-E. The hybrid thioredoxins were overexpressed in E. coli from the cloned chimeric thioredoxin genes by a T7 promoter/polymerase system. To investigate the structure-function relationship of thioredoxin, we purified the E-C3 hybrid thioredoxin through ammonium sulfate fractionation, DEAE-cellulose chromatography, and Sephadex G-50 gel filtration. Its purity was examined on SDS-polyacrylamide gel electrophoresis and the molecular weight of the purified E-C3 hybrid thioredoxin was estimated to be 12,000. On native polyacrylamide gels, the purified E-C3 hybrid thioredoxin shows a much lower mobility than E. coli thioredoxin. E-C3 hybrid thioredoxin exhibits a 40-fold lower catalytic efficiency with E. coli thioredoxin reductase than E. coli thioredoxin. It was shown to catalyze the reduction of insulin disulfide by dithiothreitol. The purified E-C3 hybrid thioredoxin was also characterized in other aspects.

• BMB Reports
• /
• 제28권2호
• /
• pp.149-154
• /
• 1995

Protein methylase II (S-adenosyl-L-methionine : protein O-methyl-transferase, EC 2.1.1.24; PM II) was purified approximately 1250-fold from porcine testis by fractional precipitation and DEAE-cellulose chromatography, followed by gel filtration on a Sephadex G-75 column and HPLC on a Protein Pak 125 column. The molecular weight of the enzyme was estimated to be 33,000 daltons by SDS-PAGE, which agreed with the value determined by gel filtration. Isoelectric focusing of purified PM II showed a single protein species with an isoelectric point of 6.2. The optimum pH for the reaction was 6.0. The $K_m$ value of the enzyme was $1{\times}10^{-5}M$ with a $V_{max}$ value of 769 pmol/min/mg of enzyme. S-adenosyl-L-homocysteine is a competitive inhibitor of PM II with a $K_i$ value of $1.38{\times}10^{-6}M$.