• 약학회지
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• 제51권5호
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• pp.291-295
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• 2007

The present study examines the effect of dominant-negative Akt on the insulin-mediated microsomal epoxide hydrolase (mEH) induction in rat hepatocytes. We also assessed the role of insulin in the expression of soluble epoxide hydrrolase (sEH). Insulin increased mEH levels and the enzyme activities, whereas sEH protein expression was unaffected by insulin. The specific PI3K inhibitors or p70 S6 kinase inhibitor ameliorated the insulin-mediated increase in mEH protein levels. Infection with adenovirus expressing dominant-negative and kinase-dead mutant of Akt1 effectively inhibited the insulin-mediated increase in mEH expression and mEH activity. These results suggest that mEH and sEH are differentially regulated by insulin and PI3K/Akt/p70S6K are active in the insulin-mediated regulation of mEH expression.

• Journal of Microbiology and Biotechnology
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• 제32권11호
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• pp.1471-1478
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• 2022

2'-Fucosyllactose (2'-FL), the most abundant fucosylated oligosaccharide in human milk, has multiple beneficial effects on human health. However, its biosynthesis by metabolically engineered Escherichia coli is often hampered owing to the insolubility and instability of α-1,2-fucosyltransferase (the rate-limiting enzyme). In this study, we aimed to enhance 2'-FL production by increasing the expression of soluble α-1,2-fucosyltransferase from Helicobacter pylori (FucT2). Because structural information regarding FucT2 has not been unveiled, we decided to improve the expression of soluble FucT2 in E. coli via directed evolution using a protein solubility biosensor that links protein solubility to antimicrobial resistance. For such a system to be viable, the activity of kanamycin resistance protein (Kan^R) should be dependent on FucT2 solubility. Kan^R was fused to the C-terminus of mutant libraries of FucT2, which were generated using a combination of error-prone PCR and DNA shuffling. Notably, one round of the directed evolution process, which consisted of mutant library generation and selection based on kanamycin resistance, resulted in a significant increase in the expression level of soluble FucT2. As a result, a batch fermentation with the ΔL M15 pBCGW strain, expressing the FucT2 mutant (F#1-5) isolated from the first round of the directed evolution process, resulted in the production of 0.31 g/l 2'-FL with a yield of 0.22 g 2'-FL/g lactose, showing 1.72- and 1.51-fold increase in the titer and yield, respectively, compared to those of the control strain. The simple and powerful method developed in this study could be applied to enhance the solubility of other unstable enzymes.

• Korean Chemical Engineering Research
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• 제47권5호
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• pp.624-629
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• 2009

분자진화방법을 이용하여 불용성 단백질을 가용성 단백질로 개량하고자 할 때 가장 중요한 과정은 발현 단백질의 세포 내 폴딩 및 용해도를 어떻게 측정하고 선별할 수 있는가에 있다. 본 연구에서는 ampicillin에 저항성을 가지는 beta-lactamase를 목적 단백질과 접합 형태로 발현하여 목적 단백질의 용해도를 측정 및 선별할 수 있는 방법을 구축하였다. 이를 위하여 먼저 beta-lactamase C-말단에 목적 단백질을 링커를 이용하여 접합단백질 형태로 발현시킬 수 있는 발현 시스템을 구축하였고, 구축된 발현시스템이 대장균의 ampicillin의 저항성을 향상시킴을 확인하였다. 구축된 발현시스템에 용해도가 비교적 높은 adenine deaminase와 aspartate aminotranseferase, 용해도가 매우 낮은 GlcNAc-2-epimerase 세가지 단백질의 유전자를 클로닝하여 Ampicillin 농도에 따라 목적 단백질의 용해도가 세포 성장에 미치는 영향을 조사하였다. Ampicillin 농도 $200{\mu}g/mL$에서 가용성 단백질인 adenine deaminase와 aspartate aminotranseferase의 접합 단백질 발현은 세포 성장을 보이는 반면, 불용성 단백질인 GlcNAc-2-epimerase 접합 단백질 발현은 세포 성장을 저해함을 확인하였다.

• Parasites, Hosts and Diseases
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• 제50권1호
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• pp.29-35
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• 2012

The aim of the study is to characterize the phenotypes of $CD4^+$ $CD25^+$ T regulatory cells within the liver granulomas and association with both Foxp-3 gene expression and splenic cytokines. Naive C57BL/6 mice were intravenously injected with multiple doses of the soluble egg antigen (SEA) 7 days before cercarial infection. The immunized and infected control groups were sacrificed 8 and 16 weeks post-infection (PI). Histopathology, parasitological parameters, splenic phenotypes for T regulatory cells, the FOXP-3 expression in hepatic granuloma using real-time PCR, and the associated splenic cytokines were studied. Histopathological examination of the liver revealed remarkable increase in degenerated ova within hepatic granuloma which decreased in diameter at weeks 8 and 16 PI ($P$<0.01). The percentage of T regulatory cells ($CD4^+$ $CD25^+$) increased significantly ($P$<0.01) in the immunized group compared to the infected control at weeks 8 and 16 PI. The FOXP-3 expression in hepatic granulomas increased from 10 at week 8 to 30 fold at week 16 PI in the infected control group. However, its expression in the immunized group showed an increase from 30 at week 8 to 70 fold at week 16 PI. The splenic cytokine levels of pro-inflammatory cytokines, IFN-${\gamma}$, IL-4, and TNF-${\alpha}$, showed significant decreases ($P$<0.05) compared to the infected control group. In conclusion, the magnitude and phenotype of the egg-induced effects on T helper responses were found to be controlled by a parallel response within the T regulatory population which provides protection in worm parasite-induced immunopathology.

• 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 and Biotechnology
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• 제9권3호
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• pp.376-380
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• 1999

Constructs, encoding a single-chain variable fragment of a catalytic antibody 4f4f (scFv-4f4f) with glycosidase activity, were made by combining the coding sequences for the heavy and light chain variable domains with a sequence encoding a linker (GGGGS). Using three different plasmid systems, single-chain antibodies were expressed separately in Escherichia coli, demonstrating significant differences in the expression level and amounts in soluble form of the recombinant protein. The protein expression from pET3a-scFv-4f4f was up to 20% of the total soluble proteins and, more importantly, the proteins were mostly found in a soluble form. An SDS-PAGE analysis of the purified single-chain proteins, yielding higher than 5mg from a 1-1 culture, showed a single band corresponding to its molecular weight of 29,100. A preliminary study shows that the expressed scFv-4f4f is catalytically active. The catalytic parameters for the hydrolysis of p-nitrophenyl-$\beta$-D-glucopyranoside by scFv-4f4f are being investigated.

• Molecules and Cells
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• 제26권1호
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• pp.34-40
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• 2008

To express an increased level of recombinant Mefp1 (marine mussel adhesive protein) in soluble form, we constructed expression vectors encoding truncated OmpA signal peptide-Mefp1 fusion proteins. OmpA signal peptide (OmpASP) is the 21 residue peptide fragment of the 23 residue OmpA signal sequence cleavable by signal peptidase I. We successfully produced increased levels of soluble recombinant Mefp1 (rMefp1) with various deletions of OmpASP, and found that the increased expression was caused by the increased pI of the N-terminus of the fusion proteins (${\geq}10.55$). All the OmpA signal peptide segments of 3-21 amino acids in length had the same pI value (10.55). Our results suggest that the pI value of the truncated OmpASP ($OmpASP_{tr}$) play an important role in directional signaling for the fusion protein, but we found no evidence for the presence of a secretion enhancer in OmpASP. For practical applications, we increased the expression of soluble rMefp1 with $OmpASP_{tr}$ peptides as directional signals, and obtained rMefp1 with the native amino terminus (nN-rMefp1) using an $OmpASP_{tr}$ Xa leader sequence that contains the recognition site for Xa protease.

• 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.

• 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.