• KSBB Journal
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• 제23권3호
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• pp.205-212
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• 2008

본 연구의 목적은 위치특이적 펩타이드 및 단백질을 사용하여 재조합 대장균에서 활성형 인간 상피세포성장인자(hEGF)를 고효율로 발현할 수 있는 방법을 찾아내는 데 있다. 재조합 대장균내 cytoplasm 및 periplasm 영역에서 hEGF의 발현을 위해 각각 세개의 응합 펩타이드 및 단백질을 선정하여 상호 비교하였다. 재조합 대장균에서 hEGF의 발현유도시 대부분 불용성 단백질로 생산되는 현상을 극복하기 위해 cytoplasm영역에서는 ATS, thioredoxin, 리파제를 융합파트너로 사용하였으며 periplasm 영역에서는 foldase인 DsbA와 DsbC, 용융성 고발현 단백질인 maltose binding protein을 선택하여 사용하였다. Periplasm영역에서 발현유도를 시키는 융합단백질의 경우 cytoplasm영역에서의 발현양도 용융성 형태로 고발현 되는 것을 알 수 있었으며 전체적으로 약 2배가량의 용융성 형태로 발현되었다. hECF의 발현율을 가장 높일 수 있는 융합단백 질은 maltose binding protein이었으나 발현된 융합단백질의 24%가 불용성 단백질로 형성되어 활성형 형태로 얻는 데 한계가 있었으며, 활성형 형태로 hEGF의 발현을 위해서는 DsbA를 응합단백질로 사용한 경우에 18.1 mg/L로 가장 높은 발현농도를 보였다. Cytoplasm 영역에서 발현유도를 한 경우에는 ATS와 thioredoxin을 응합파트너로 hEGF를 발현한 경우 용융성 형태로 높은 발현율을 보였다. 특히 ATS와 같은 펩타이드를 N-말단에 융합시킨 경우 불용성을 방지하는 효과를 보여 이황화결합의 불완전성이나 소수성으로 인해 불용성 단백질로 발현되는 기존의 단백질을 활성형 형태로 발현하는데 될 수 있음을 확인할 수 있었다.

• 한국축산식품학회지
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• 제35권5호
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• pp.707-713
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• 2015

Royal jelly has been widely used as a health supplement worldwide. However, royal jelly has been implicated in allergic reactions, and we developed a water-soluble royal jelly (WSRJ) without the allergy inducing protein. In this study, we aimed to identify the anti-melanogenic efficacy of WSRJ. B16F1 melanoma cells were first treated with 10 nM α-melanocyte stimulating hormone (α-MSH) and then with various doses of WSRJ. In addition, we investigated the mRNA and protein expression of melanogenesis-related genes such as tyrosinase, tyrosinase related protein-1 (TRP-1) and TRP-2 by reverse transcription-polymerase chain reaction and western blotting. WSRJ directly inhibited tyrosinase and cellular tyrosinase activity, which decreased melanin synthesis in α-MSH stimulated B16F1 melanoma cells a level comparable to that observed with arbutin. WSRJ decreased the mRNA and protein expressions of tyrosinase, TRP-1, and TRP-2, which was comparable to that observed with arbutin. WSRJ has strong anti-melanogenic activity, which invoice direct inhibition of tyrosinase enzyme activity and suppression of expression of melanogenesis related genes. Results from this study suggests that WSRJ is a potential candidate for the treatment of skin pigmentation.

• 한국약용작물학회지
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• 제17권4호
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• pp.293-300
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• 2009

This study was carried out to investigate the effect of Cinnamomum camphora on melanogenesis. The MeOH extract of Cinnamomum camphora inhibited mushroom tyrosinase activity in dose-dependent manner. Moreover, it significantly suppressed the melanin production in melan-a cells at the concentration of $100{\mu}/m{\ell}$. The MeOH extract was partitioned with ethyl acetate, n-butanol and water. Among them, the BuOH soluble fraction exhibited significant inhibitory effect on mushroom tyrosinase. In addition, the BuOH soluble fraction reduced the melanin production in melan-a cells. But, the BuOH soluble fraction had less inhibition effects on melan-a cell originated tyrosinase. So, it was performed western blotting for melanogenic proteins (tyrosinase, tyrosinase-related protein (TRP-2)) using melan-a cells. The BuOH soluble fraction inhibited the protein expression of tyrosinase at the concentration of $100{\mu}/m{\ell}$. The results suggested that the BuOH soluble fraction of C. camphora might be a potent inhibitor of melanin biosynthesis in melan-a cells.

• 수산해양교육연구
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• 제28권4호
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• pp.903-912
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• 2016

Hagfish which belongs to the chordate contact cyclostomata, is important phylogenetic relationship between vertebrate and invertebrate. Cathepsins of the cysteine protease family have traditionally been thought to play a major role in intracellular protein degradation and turnover in lysosomes. In this study, Catepsin L was cloned from Inshore hagfish (Eptatretus burgeri), the cDNA encoding ORF of the Eptatretus burgeri Cathepsin L (EbCtL) is 978 bp. The cDNA encoding proEbCtL was expressed in Escherichia coli strain BL21(DE3) using the pGEX-4T-1 expression vector system. The recombinant proEbCtL protein was overexpressed as a approximately 55 kDa fusion protein. The overproduced soluble GST-fusion protein was then applied to glutathione-Sepharose 4B column chromatography; the sample harboring the fusion protein evidenced a high degree of purity when analyzed via SDS-PAGE and Western blot analysis. Its activity was quantied by cleaving the synthetic peptide Z-FR-AMC, Z-LLE-AMC, and Suc-AAF-AMC, and the optimal pH for the protease activity was 8, 9.5, and 9, respectively.

• Journal of Microbiology and Biotechnology
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• 제18권5호
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• pp.845-851
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• 2008

TolC is an outer membrane porin protein and an essential component of drug efflux and type-I secretion systems in Gram-negative bacteria. TolC comprises a periplasmic $\alpha$-helical barrel domain and a membrane-embedded $\beta$-barrel domain. TdeA, a functional and structural homolog of TolC, is required for toxin and drug export in the pathogenic oral bacterium Actinobacillus actinomycetemcomitans. Here, we report the expression of the periplasmic domain of TdeA as a soluble protein by substitution of the membrane-embedded domain with short linkers, which enabled us to purify the protein in the absence of detergent. We confirmed the structural integrity of the TdeA periplasmic domain by size-exclusion chromatography, circular dichroism spectroscopy, and electron microscopy, which together showed that the periplasmic domain of the TolC protein family fold correctly on its own. We further demonstrated that the periplasmic domain of TdeA interacts with peptidoglycans of the bacterial cell wall, which supports the idea that completely folded TolC family proteins traverse the peptidoglycan layer to interact with inner membrane transporters.

• 생명과학회지
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• 제12권1호
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• pp.113-119
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• 2002

방선균에 존재하는 단백질 타이로신 포스파타아제의 기능을 알아보기 위하여 우선 이 유전자를 대장균에 클로닝하여 대량으로 발현시킨 결과, 발현된 단백질은 soluble형태로 존재하여 자신의 효소활성을 가지고 있었으나, 효소활성부위에 대한 변이주의 경우에는 기질과 결합은 하였으나 활성이 없는 것으로 나타났었다. 방선균에서 이 효소의 세포내 기작 및 결합 단백질을 파악하기 위해 대장균에 클로닝한 유전자를 방선균 발현벡터(pIJ6021)에 클로닝을 하였다. 이렇게 만든 유전자를 in-ducer인 thiostrepton을 이용하여 발현시킨 결과, 활성형의 단백질 타이로신 포스파타아제가 대량으로 생산되었다. 그리고 이들 유전자를 방선균에서 과잉 발현시킨 결과 항생제 생산 및 형태 변화 등의 표현형은 나타나지 않았다. 이효소와 반응하는 기질 및 결합 단백질을 찾기 위해서 이들과 결합은 하지만 반응하지 않는 돌연변이 단백질 타이로신 포스파타아제 (PtpA(C9S))를 유전자 조작하여 방선균에서 과잉 발현시켰다 그 결과 표현형은 없었지만 인산 타이로신 단백질의 패턴변화를 알 수 있었고, 단백질 타이로신 포스파타아제에 의하여 인산화 조절되는 단백질 3개 찾을 수 있었다. 이들 세 단백질(p65, p90 그리고 p200)은 ptpA(C9S)가 발현된 방선균에서 보다 더 많은 인산화 패턴을 나타내었으며, 이들은 가능한 단백질 타이로신 포스파타아제의 표적이라고 생각되었다. 만약 이들의 구조가 밝혀진다면, 방선균의 타이로신 포스파타아제의 기능 및 신호전달 과정의 역할을 파악할 수 있으리라 생각된다.

• Journal of Plant Biotechnology
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• 제1권1호
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• pp.46-55
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• 1999

The expressed sequence tags(ESTs) from immature seed of rice, Oryza sativa cv Milyang 23, were partially sequenced and analyzed by homology. As of 1998, the partial sequences of about 6,600 cDNA clones were analyzed from normal and normalized immature seed cDNA libraries. About 2,200 ESTs were putatively identified by BLASTX deduced amino acid sequence homology analysis. About 20% of them were putatively identified as storage proteins. Also the clones were highly homologous to genes involved particularly in starch biosynthesis, glycolysis, signal transduction and defenses. Compared to 35% of redundancy in the ESTs of normal cDNA library, that from the substracted library was 15%. The Korea Rice Genome Network is maintained to provide the updated information of sequences, their homologies and sequence alignments of ESTs. For the stable expression of transgene in rice, diverse vectors were developed for overexpression, targeting and gene dosage effect with transit peptides (Tp) and matrix attachment region (MAR) sequence from chicken lysozyme locus. The rice calli were transformed via Agrobacterium tumefaciens LBA4404(pSB1) with the triparental mating technique and selected by herbicide resistance. The green fluorescent protein(GFP) gene in expression vector under the control of rbcS promoter-Tp was overexpressed upto 10 % of the total soluble protein. In addition, the Tp-sGFP fusion protein was properly processed during translocation into chloroplast. The expression of sGFP in the presence of MAR sequences was analyzed with Northern and immunoblot analysis. All the lines in which sGFP transgene with MAR sequence, showed position independent and copy number-dependent expression, while the lines without MAR showed the varied level of expression with the integration site. Thus the MAR sequence significantly reduced the variation in transgene expression between independent transformants.

• Journal of Periodontal and Implant Science
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• 제32권4호
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• pp.721-731
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• 2002

Recently, soluble TNF receptor homolog osteoprotegerin(OPG) and its membrane-bound ligand osteoclast differentiation factor(ODF) were found to regulate osteoclast formation and function, and bone metabolism. It is now well established that ODF acts via RANK expressed on hematopoietic osteoclast precursor cells to facilitate their differentiation to osteoclasts, and OPG prevents the formation of osteoclasts by interfering the binding of ODF and RANK. Expression of OPG and ODF was believed to be closely related to the pathogenesis of bone resorption and destruction from osteoporosis, periodontal diseases, malignant bone tumor, and arthritis. The periodontal ligament fibroblasts (PDLF), located between the tooth and tooth socket, has been thought to play an important role in maintaining bone homeostasis of periodontal tissues. However, the exact mechanism by which bone formation and resorption are regulated by PDLF is not well understood. In this study we have prepared primary cultures of human PDLF from periodontium of malaligned tooth extracted due to orthodontic reason, and determined steady state or inflammatory signal-induced OPG and ODF expression using RT-PCR and western blot analysis. OPG and ODF mRNA and protein were expressed constitutively in the PDLF and these expression were slightly increased by osteotropic cytokine IL-1 ${\beta}$. Lipopolysaccharide-treated PDLF showed decrease in OPG mRNA and protein expression, and increase in ODF mRNA and protein expression. These results indicated that PDLF influence the osteoclastogenesis by OPG and ODF expression in the inflammatory situation as well as physiological condition, and thereby pathogenesis of periodontal alveolar bone destruction.

• Asian Pacific Journal of Cancer Prevention
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• 제14권10호
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• pp.5651-5655
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• 2013

Objective: Major histocompatibility complex class I chain-related A (MICA) is a stress-inducible glycoprotein that can be shed as a soluble protein. This study was conducted to determine the expression of MICA in renal cell carcinoma (RCC) and examine the clinical relevance of soluble MICA (sMICA) in this disease. Methods: Immunohistochemistry and real-time PCR analyses were performed to assess the expression of MICA in 48 pairs of RCC and adjacent normal renal tissues. Serum levels of sMICA were measured in 48 RCC patients, 12 patients with benign renal tumors, and 20 healthy individuals. The correlations between sMICA levels and clinicopathological parameters were analyzed and the diagnostic performance of sMICA in RCC was evaluated. Results: RCCs exhibited elevated expression of MICA compared to adjacent normal tissues. Serum concentrations of sMICA were significantly greater in RCC patients ($348.5{\pm}32.5pg/ml$) than those with benign disease ($289.3{\pm}30.4pg/ml$) and healthy controls ($168.4{\pm}43.2pg/ml$) and significantly correlated with advanced tumor stage, lymph node metastasis, distant metastasis, vascular invasion, and higher histological grade. Using a cut-off point of 250 pg/ml, sMICA demonstrated a specificity and sensitivity of 63.2% and 75.6%, respectively, in distinguishing between RCC and benign renal tumors. Conclusion: MICA expression is upregulated in RCC and increased serum sMICA levels predict aggressive tumor behavior. However, the applicability of sMICA alone is limited in distinguishing RCC from benign renal tumors.

• Journal of Microbiology
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• 제44권3호
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• pp.293-300
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• 2006

The Escherichia coli heat-labile enterotoxin B subunit (HLT-B) is one of the most powerful mucosal immunogens and known mucosal adjuvants. However, the induction of high levels of HLT-B expression in E. coli has proven a difficult proposition. Therefore, in this study, the HLT-B gene was cloned from pathogenic E. coli and expressed as a fusion protein with GST (glutathion S-transferase) in E. coli BL2l (DE3), in an attempt to harvest a large quantity of soluble HLT-B. The culture conditions, including the culture media used, temperature, pH and the presence of lactose as an inducer, were all optimized in order to obtain an increase in the expression of soluble GST-rHLT-B. The biological activity of the purified rHLT-B was assayed in a series of GMI-ELISA experiments. The findings of these trials indicated that the yield of soluble recombinant GST-rHLT-B could be increased by up to 3-fold, as compared with that seen prior to the optimization, and that lactose was a more efficient alternative inducer than IPTG. The production of rHLT-B, at 92 % purity, reached an optimal level of 96 mg/l in a 3.7 L fermentor. The specific GM1 binding ability of the purified rHLT-B was determined to be almost identical to that of standard CTB.