• Journal of Microbiology and Biotechnology
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• 제25권2호
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• pp.274-279
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• 2015

Receptor activator of nuclear factor-kappa B ligand (RANKL) is a critical factor in osteoclastogenesis. It makes osteoclasts differentiate and multinucleate in bone remodeling. In the present study, RANKL was expressed as a soluble maltose binding protein (MBP)-fusion protein using the Escherichia coli maltose binding domain tag system (pMAL) expression vector system. The host cell E. coli DH5α was cultured and induced by isopropyl β-_D-1-thiogalactopyranoside for rRANKL expression. Cells were disrupted by sonication to collect soluble MBP-fused rRANKL. The MBP-fusion rRANKL was purified with MBP Trap affinity chromatography and treated with Tobacco Etch Virus nuclear inclusion endopeptidase (TEV protease) to remove the MBP fusion protein. Dialysis was then carried out to remove binding maltose from the cleaved rRANKL solution. The cleaved rRANKL was purified with a second MBP Trap affinity chromatography to separate unsevered MBP-fusion rRANKL and cleaved MBP fusion protein. The purified rRANKL was shown to have biological activity by performing in vitro cell tests. In conclusion, biologically active rRANKL was successfully purified by a simple two-step chromatography purification process with one column.

• 미생물학회지
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• 제31권4호
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• pp.274-278
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• 1993

단백질이 어떻게 비수용성이 되는지를 알기위해, 클로람페니콜 아세틸전이효소와 베타-락타메이즈를 과잉생산하여 그들의 수용성과 활성을 측정하였다. 클로람페니콜 아세틸전이효소는 총단백질의 9에서 45%를 차지하였으며, inclusion body 형성없이 완전히 수용성이었으며, 효소활성은 만들어진 양과 비례하였다. 또한 30℃에서 T7 발현체계에 의해 생성된 베타-락타메이즈는 수용성의 숙성체였으나, 37℃에서는 비수용성이 되었다. 세포질에 있는 대부분의 베타-락타메이즈는 비수용성이었고. 페리플라즘 공간에서는 대부분이 수용성이었다. 단백질의 올바른 폴딩을 도와주는 chaperone의 일종인 GroEL 단백질은 본 실험조선에서는 베타-락타베이즈의 수용성을 별로 높이지는 못했다. 세포 내에서 inclusion body의 형성은 단백질의 높은 종도보다는 각각 단밸질 자체의 특성과 관련된 듯하다.

• KSBB Journal
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• 제23권5호
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• pp.403-407
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• 2008

본 연구에서는 현재 산업적 응용이 활발하게 이루어지고 있고, 여러 장점을 지닌 효소인 Candida antarctica에서 유래된 lipase B (CalB)의 신속한 개질을 위해 취급이 용이한 E. coli를 이용하여 CalB 발현시스템을 구축하였다. E. coli 발현 시스템에서 효소활성을 지니지 못하는 내포체를 생성하는 단점을 지니고 있어, soluble한 형태의 CalB 생성을 위해 저온 발현이 가능한 pCold I vector와 단백질 접힘을 도와주는 chaperone을 사용하여 CalB를 발현하였다. Liu 등(17)은 E. coli Origami2와 B, 그리고 $DH5{\alpha}$를 실험한 결과, Origami 균주에서만 CalB에 의한 halo의 형성이 관찰되었으나, 동 연구에서는 실험한 3종의 균주와 5종의 chaperone plasmid중 Rosettagami와 $DH5{\alpha}$에서 groES/groEL chaperone이 CalB와 동시에 발현되면 soluble한 형태의 Cal B가 발현됨을 관찰할 수 있었다. 또한 신속한 CalB의 발현시스템을 구축하기 위해서는 유전자 조작의 용이성 및 안정성에서 우월한 $DH5{\alpha}$가 Rosettagami에 비해 soluble한 CalB의 발현에 더욱 적합한 균주임이 관찰되었다. 즉 재조합 pCold plasmid와 pGro7 plasmid (groES/groEL)로 형질이 전환된 $DH5{\alpha}$가 CalB 발현시스템에 가장 적합하다.

• Journal of Microbiology and Biotechnology
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• 제17권4호
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• pp.638-643
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• 2007

The full encoding sequence for human type II hexokinase (HXK II) was cloned into the E. coli expression vector pET 21b and expressed as a C-terminally hexahistidine-tagged protein in the BL2l (DE3) strain. The IPTG-induced HXK II approximately accounted for 17% of the total E. coli proteins, and 81% of HXK $II_{6{\times}His}$ existed in inclusion bodies. To improve the production of soluble recombinant HXK II protein, in the functionally active form, we used low temperature, and the osmotic stress expression method. When expressed at $18^{\circ}C$, about 83% of HXK $II_{6{\times}His}$ existed in the soluble fraction, which amounted to a 4.1-fold yield over that expressed at $37^{\circ}C$. The soluble form of HXK $II_{6{\times}His}$ was also highly produced in the presence of 1M sorbitol under the standard condition $(37^{\circ}C)$, which indicated that temperature downshift and low water potentials were required to improve the yield of active recombinant HXK II protein. The expressed protein was purified by metal chelate affinity chromatography performed in an IDA Excellose column charged with $Ni^{2+}$ ions, resulting in about 40mg recombinant HXK II protein obtained with purity over 89% from 51 of E. coli culture. The identity of HXK $II_{6{\times}His}$ was confirmed by Western blotting analysis. Taken together, using the stress-governed expression described in this study, human active HXK II can be purified in sufficient amounts for biochemical and biomedical studies.

• Journal of Nutrition and Health
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• 제33권7호
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• pp.712-716
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• 2000

The Purpose of this study is to investigate the effect of soluble dietary fiber psyllium on insulin sensitivity and skeletal muscle glucose transporter 4(GLUT4) protein expression in stroke-prone hypertensive rats(SHRSP) fed a high-fat diet containing 5% of psyllium or cellulose from five to nine weeks of age. Obtained results were as follows : (1) In the psyllium diet group fasting plasma glucose level was significantly reduced and glucose levels upon oral glucose tolerance test were significantly lower than cellulose diet group at 30 min(p<0.05) and 60 min(p<0.01) (2) Skeletal muscle GLUT4 contents were significantly increased in the soleus(slow twitch) and extensor digitorum longus(fast twitch) muscle of psyllium diet group. (3) However there was no difference in insulin levels in the fasting and oral glucose tolerance test. These results indicated that psyllium diet improves peripheral insulin sensitivity but not insulin secretion. In conclusion our present finding suggest that soluble fiber diet is effective to increase insulin sensitivity in SHRSP. From these results it was suggested that soluble dietary fiber supplementation effectively prevents insulin resistance.

• Biotechnology and Bioprocess Engineering:BBE
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• 제11권5호
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• pp.414-419
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• 2006

When the alginate lyase gene (aly) from Pseudoalteromonas elyakovii was expressed in E. coli, most of the gene product was organized as aggregated insoluble particles known as inclusion bodies. To examine the effects of chaperones on soluble and nonaggregated form of alginate lyase in E. coli, we constructed plasm ids designed to permit the coexpression of aly and the DnaK/DnaJ/GrpE or GroEL/ES chaperones. The results indicate that coexpression of aly with the DnaK/DnaJ/GrpE chaperone together had a marked effect on the yield alginate lyase as a soluble and active form of the enzyme. It is speculated this result occurs through facilitation of the correct folding of the protein. The optimal concentration of L-arabinose required for the induction of the DnaK/DnaJ/GrpE chaperone was found to be 0.05mg/mL. An analysis of the protein bands on SDS-PAGE gel indicated that at least 37% of total alginate lyase was produced in the soluble fraction when the DnaK/DnaJ/GrpE chaperone was coexpressed.

• 생약학회지
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• 제48권4호
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• pp.314-319
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• 2017

To evaluate the longevity property of Allium hookeri (Liliaceae) leaves, this study was performed. Ethanol extract of A. hookeri was successively partitioned as methylene chloride, ethyl acetate, n-butanol and $H_2O$ soluble fractions, and lifespan-extending effects of the fractions were checked using Caenorhabditis elegans model system. The most active ethyl acetate-soluble fraction elevated heat stress tolerance, and increased expression of stress resistance protein. Further studies were performed to investigate several aging-related factors such as reproduction, food intake, growth and movement of C. elegans. The results revealed that there were no significant changes in aging-related factors including reproduction and food intake, however, ethyl acetate-soluble fraction treatment led to up-regulation of locomotory ability and growth of aged worms, suggesting ethyl acetate-soluble fraction affected healthspan as well as lifespan of nematode.

• KSBB Journal
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• 제16권1호
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• pp.1-10
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• 2001

Escherichia coli has been used as an expression work horse for foreign genes. This article summarized recent development in genetic engineering techniques for overproduction of medical proteins and industrial enzymes. Special emphasis was placed upon research activities concerning folding and refolding of inclusion bodies at genetic and fermentation levels. Plasmid and mRNA stabilization, development of strong inducible promoters, modification of translational elements and reduction of rpoteolytic degradation were carried out to elevate an expression level of a target protein. Optimization of culture conditions, improvement of denaturation and renaturation steps and coexpression of molecular chaperones or foldase were accomplished to produce active proteins in soluble form. Fusion protein systems with selective separation and surface display technology were also performed in an effort to make the E. coli expression system more effective and versatile.

• The Korean Journal of Physiology and Pharmacology
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• 제4권5호
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• pp.379-383
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• 2000

Pathophysiological implications of the vascular nitric oxide (NO)/cGMP pathway in hypertension were investigated. Sprague-Dawley rats were made deoxycorticosterone acetate (DOCA)-salt hypertensive for six weeks. The protein expression of endothelial constitutive NO synthase (ecNOS) and the tissue content of NO were determined in the thoracic aorta. The protein expression and catalytic activity of soluble guanylyl cyclase (GC) were also determined. Systolic blood pressure measured on the day of experiment was significantly higher in the experimental group than in the control. The hypertension was associated with decreases in the vascular tissue content of NO metabolites, concomitantly with the expression of ecNOS proteins. The protein expression of GC was not affected, while its catalytic activity was significantly decreased in hypertension. These results indicate that the high blood pressure is associated with a decreased activity of vascular NO/cGMP pathway in DOCA-salt hypertension.

• 한국자기공명학회논문지
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• 제17권1호
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• pp.30-39
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• 2013

Immortalization-upregulated protein-1 (IMUP-1) genes have been cloned and are known to be involved in SV40-mediated immortalization. IMUP-1 gene is highly expressed in various cancer cell lines and tumors, suggesting the possibility that they might be involved in tumorigenicity. Previously, there were several problems for overexpression of IMUP-1 in bacterial expression systems including low solubility and aggregation due to unstructured property. To investigate the structural properties, it is necessary to obtain lots of pure and soluble proteins. Accordingly, the co-expression systems of bacterial chaperone proteins, GroEL-GroES, were used to increase solubility of IMUP-1. From the analysis of NMR and CD experiment data, it is suggested that the protein adopt typical the random coil properties in solution.