• Journal of Microbiology and Biotechnology
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• 제25권8호
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• pp.1227-1233
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• 2015

Vascular endothelial growth factor (VEGF) plays a key role in angiogenesis through binding to its specific receptors, which mainly occurs to VEGF receptor 2 (VEGFR-2), a kinase insert domain-containing receptor. Therefore, the disruption of VEGFR-2 signaling provides a promising therapeutic approach for the treatment of cancer by inhibiting abnormal or tumorinduced angiogenesis. To explore this potential, we expressed the catalytic domain of VEGFR-2 (VEGFR-2-CD) as a soluble active kinase in Escherichia coli. The recombinant protein was purified and the VEGFR-2-CD activity was investigated. The obtained VEGFR-2-CD showed autophosphorylation activity and phosphate transfer activity comparable to the commercial enzyme. Furthermore, the IC₅₀ value of known VEGFR-2 inhibitor was determined using the purified VEGFR-2-CD. These results indicated a possibility for functional and economical VEGFR-2-CD expression in E. coli to use for inhibitor screening.

• 대한약학회:학술대회논문집
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• 대한약학회 2003년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.2-2
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• pp.160.1-160.1
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• 2003

In order to confirm the biological function of ORF10 in cephabacin biosynthesis gene cluster of Lysobacter lactamgenus as an ATP-binding-cassette (ABC) transporter, the gene for ORF10 was amplified and subcloned into pET-28a(+) expression vector. After gene induction with 0.5 mM IPTG at 30~! and further cultivation at $30^~$ !. for 8 hr, a lot of the recombinant ORF10 protein was produced as soluble form in cytoplasmic fraction as well as a membrane protein in the membrane fraction as likely as other ABC transporters. (omitted)

• Journal of Microbiology and Biotechnology
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• 제17권7호
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• pp.1113-1119
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• 2007

Human bone marrow-derived mesenchymal stem cells (hBMMSCs) must differentiate into osteogenic cells to allow for successful bone regeneration. In this study, we investigated the effects of different combinations of three soluble osteogenic differentiation-inducing factors [L-ascorbic acid (AC), ${\beta}$-glycerophosphate (${\beta}G$), and bone morphogenic protein-2 (BMP-2)] and the presence of a hydroxyapatite (HA) substrate on hBMMSC osteogenic differentiation in vitro. hBMMSCs were cultured in medium containing various combinations of the soluble factors on culture plates with or without HA coating. After 7 days of culture, alkaline phosphatase (ALP) activity, calcium deposition, and osteoprotegerin (OPG) and osteopontin (OPN) expression were measured. The effects of individual and combined factors were evaluated using a factorial analysis method. BMP-2 predominantly affected expression of early markers of osteogenic differentiation (ALP and OPG). HA had the highest positive effect on OPN expression and calcium deposition. The interaction between AC, ${\beta}G$, and HA had the second highest positive effect on ALP activity.

• Journal of Microbiology and Biotechnology
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• 제19권12호
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• pp.1620-1627
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• 2009

The design and expression of an antihypertensive peptide multimer (AHPM), a common precursor of 11 kinds of antihypertensive peptides (AHPs) tandemly linked up according to the restriction sites of gastrointestinal proteases, were explored. The DNA fragment encoding the AHPM was chemically synthesized and cloned into expression vector pGEX-3X. After an optimum induction with IPTG, the recombinant AHPM fused with glutathione S-transferase (GST-AHPM) was expressed mostly as inclusion body in Escherichia coli BL21 and reached the maximal production, 35% of total intracellular protein. The inclusion body was washed, dissolved, and purified by cation-exchange chromatography under denaturing conditions, followed by refolding together with size-exclusion chromatography and gradual dialysis. The resulting yield of the soluble GSTAHPM (34 kDa) with a purity of 95% reached 399 mg/l culture. The release of high active fragments from the AHPM was confirmed by the simulated gastrointestinal digestion. The results suggest that the design strategy and production method of the AHPM will be useful to obtain a large quantity of recombinant AHPs at a low cost.

• Journal of Plant Biotechnology
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• 제37권4호
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• pp.556-561
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• 2010

Lactoferrin is an 80-kDa iron-binding glycoprotein that is found in high concentrations in human milk. Human lactoferrin (hLF) has several beneficial biological activities including immune system modulation and antimicrobial activity. In the present study, we devolved a method of hLF expression through introducing the hLF gene construct into Oriza sativa cv. Nakdong using the Agrobacterium-mediated transformation system. The expression of the hLF gene under the control of the rice glutelin promoter was detected in the seeds of transgenic rice plants. Transformed rice plants were selected on media containing herbicide(DL-phosphinothricin) and the integration of hLF cDNA was confirmed by Southern blot analysis. The expression of the full length hLF protein from the grains of transgenic rice plants was verified by Western blot analysis. The lactoferrin expression levels in the transformed rice grains determined by enzyme-linked immunosorbant assay accounted for approximately 1.5% of total soluble protein. Taken together, these data indicate that rice grains expressing hLF can be directly incorporated into infant formula and baby food.

• 생명과학회지
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• 제14권1호
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• pp.121-125
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• 2004

E. coli에서 B. macerans 유래 cyclodextrin glucanotransferase (CGTase)의 활성형 생산에 GroEL/ES chaperone과의 공발현과 저온 배양의 공동작용 효과에 대해 조사하였다. 실험에 사용된 cgt와 groEL/ES 유전자를 발현하는 pTCGTl과 pGroll은 각각 T7 promoter와 Pzt-1 promoter에 의해 조절되고 이들을 E. coli에 도입시켰다. 대수증식기 초기(2 hr)와 대수증식기 중기(3 hr)에 tetrarycline 10 ng/ml 과 IPTG 1 mM을 첨가하여 각각의 유전자를 발현시켰다. CGTase활성과 specific artivity 측정 시 $37^{\circ}C$에서 pTCGTl 단독 발현 보다 $25^{\circ}C$에서 chaperone과 함께 발현시킨 경우 2배나 높은 활성이 측정됐으며, SDS-PACE 분석결과 $37^{\circ}C$에서 단독 발현 시킬경우 20% 정도 가용성 형태로 발현되던 것이 $25^{\circ}C$에서 chaperone과 공발현 시에는 거의 3.5배가 넘는 69%가 가용성으로 전환됨을 알 수 있었다. 이와 같이 분자 chaperone과 $25^{\circ}C$에서의 저온 배양은 E. coli에서 활성형질 가용성 CGTase의 생산 증가에 큰 영향을 미치는 것으로 나타났다.

• Journal of Plant Biotechnology
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• 제7권2호
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• pp.97-103
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• 2005

This study was carried out to obtain basic information for gene manipulation in potent edible tobacco (Nicotiana tabacum cv. TI 516). N. tabacum cv. TI 516 is a plant for a possible candidate to use as an edible vaccine, since it contains a low level of nicotine. The effective plant regeneration system through leaf disc culture was achieved using a MS basal medium supplemented with 0.1 mg $1^{-1}$ NAA and 0.5 mg $1^{-1}$ BA. In order to transform the N. tabacum cv. TI 516 with the green fluorescent protein (GFP) gene, Agrobacterium tumefaciens LBA 4404 containing the GFP gene was used. Genomic PCR confirmed the integration of the GFP gene into nuclear genome of transgenic plants. Expression of the GFP gene was identified in callus, apical meristem and root tissue of transgenic N. tabacum cv. TI 516 plants using fluorescence microscopy. Western blot analysis revealed the expression of GFP protein in the transgenic edible tobacco plants. The amount of GFP protein detected in the transgenic tobacco plants was approximately 0.16% of the total soluble plant protein (TSP), which was determined by ELISA.

• 융합정보논문지
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• 제11권11호
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• pp.248-255
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• 2021

본 연구는 만성폐쇄성폐질환(COPD)의 동물 모델을 이용하여 Celecoxib의 폐 손상 개선효과를 연구하였다. COPD는 LPS와 담배연기추출물(CSE)로 유도하여 in vitro와 in vivo에서 병행 연구하였다. In vitro는 인간 섬유아세포(MRC5)에서 MTT assay, real-time PCR를 하였고 in vivo는 mRNA 발현, 기관지폐포세척액(BALF), collagen content, 단백질 발현을 확인하였다. 실험을 통해 Celecoxib는 BALF에서 염증세포 수의 감소와 사이토카인, soluble protein의 축적을 감소시켰고 동물모델에서는 체중과 폐 무게를 감소시켰으며, 폐 콜라젠 축적도 개선하였다. 또 웨스턴 블로팅과 real-time PCR을 통해 EMT 표지자의 감소를 확인하였다. 결과적으로 Celecoxib는 EMT를 조절하여 LPS+CSE로 유도된 COPD의 폐 손상에서 개선제로 작용할 수 있을 것으로 사료된다.

• Korean Chemical Engineering Research
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• 제56권5호
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• pp.711-717
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• 2018

상피세포 성장인자(Epidermal Growth Factor, EGF)는 세포 분열을 자극하고 의약적 용도가 다양하다. EGF는 3개의 이황화 결합을 갖고 불용성으로, 대장균에서 고효율 발현에 대한 연구가 잘 이루어지지 않았다. EGF 유전자를 작은 유비퀴틴 관련 유전자(small ubiquitin-related modifier gene, SUMO)와 결합하고 DE3 대장균에서 발현시켰다. IPTG (Isopropyl-${\beta}$-D-Thiogalactopyranoside)로 유도하여 대장균 세포 단백질의 38.9%로 융합단백질이 발현되었고, Ni-NTA 친화성 크로마토그래피로 분리하였다. 그 후 유비퀴틴 분해효소반응으로 융합단백질에서 EGF를 얻은 후 다시 Ni-NTA 크로마토그래피로 분리 하였다. 최종적으로 정제된 EGF의 순도는 HPLC로 분석하였으며, 98%이상의 순도를 얻을 수 있었다.

• 한국자기공명학회논문지
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• 제19권2호
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• pp.74-82
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• 2015

Ryanodine receptor (RyR) is one of the two major $Ca^{2+}$ channels in membranes of intracellular $Ca^{2+}$ stores and is found in sarcoplasmic reticulum (SR), endoplasmic reticulum (ER). RyR1 is also the major calmodulin-binding protein of sarcoplasmic reticulum membranes. Residues 4064-4210 in the RyR1 polypeptide chain has similar primary sequence with calmodulin (CaM) and was designated as CaM-like domain (CaMLD). When expressed as a recombinant peptide, CaMLD showed several CaM-like properties in previous studies. Still, previous studies of CaMLD were focused on protein-protein interactions rather than its own properties. Here, we studied the expression of CaMLD and its sub-domains corresponding to each lobe of CaM in Escherichia coli. CaMLD could be obtained only as inclusion body, and it was refolded using urea solubilization followed by dialysis. Using spectroscopic approaches, such as NMR, circular dichroism, and gel filtration experiment, we found that the refolded CaMLD exists as nonspecific aggregate, even though it has alpha helical secondary structure. In comparison, the first half of CaMLD (R4061-4141) could be obtained as natively soluble protein with thioredoxin fusion. After the removal of the fusion tag, it exhibited folded and helical properties as shown by NMR and circular dichroism experiments. Its oligomeric status was different from CaMLD, existing as dimeric form in solution. However, the second half of the protein could not be obtained as soluble protein regardless of fusion tag. Based on these results, we believe that CaMLD, although similar to CaM in sequence, has quite different physicochemical properties and that the second half of the protein renders it the aggregative properties.