• Applied Microscopy
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• 제19권2호
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• pp.74-84
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• 1989

인삼 종자의 배유조직에서 Tris 완충액 가용성 저장단백질을 추출한 후 전기 영동적 분석으로 분리하여 $SP_{1}$(MW=160,000)과 $SP_2$(MW=70,000)의 두가지 저장단백질을 정제하였다. 이 두가지 저장단백질을 항원으로 사용하여 토끼에 피하주사하여 항체를 얻었으며, 이 항체를 이용하여 면역 세포화학적 금입자표지법을 실시한 결과, $SP_1$과 $SP_2$ 모두 구형의 protein body내에 산재하여 있음을 확인되었으며, globoid에는 이러한 두가지 단백질중 어느 것도 함유되어 있지 않는 것으로 나타났다. 또한 각각의 protein body에 함유된 $SP_1$과 $SP_2$의 상대적 함량에는 서로 차이가 있음이 확인되었다.

• 한국버섯학회지
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• 제19권4호
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• pp.261-271
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• 2021

In this study, the antioxidant and anti-inflammatory effects of methanol extract (ME) and hot water extracts (HE) from the fruiting bodies of Ganoderma applanatum were investigated. The 1,1-diphenyl-2-picryl-hydrazy (DPPH) radical scavenging activity of 2.0 mg/mL ME (94.83%) was comparable to that of butylated hydroxytoluene (96.97%), the reference standard. The hydroxyl radical scavenging activities of ME and HE were similar to that of BHT at 2.0 mg/mL, whereas lipid peroxidation activity of the ME and HE were significantly lower than that of BHT. High-performance liquid chromatography analysis showed that the G. applanatum fruiting bodies contained nine phenolic compounds, which might contribute to antioxidant and anti-inflammatory activities. The survival rate of RAW 264.7 macrophages treated with 2.0 mg/mL ME and HE were 65.23 to 68.12% at 2.0 mg/mL, thereby indicating that the extracts were slightly cytotoxic at the concentration tested. The extracts also inhibited the nitric oxide (NO)-mediated expression of inducible nitric oxide synthase (iNOS) protein in lipopolysaccharide-induced RAW 264.7 macrophages and carrageenan-induced paw edema in rats. The study results demonstrated that the fruiting bodies of G. applanatum possessed good antioxidant and anti-inflammatory activities, which might be used to develop novel anti-inflammatory agents.

• Journal of Ginseng Research
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• 제15권2호
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• pp.131-138
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• 1991

This study was carried out to investigate the development of endoplasmic reticulum and the formation of Protein body in the endosperm cell during seed formation of Panax ginseng C. A. Meyer with electron microscope. In the endosperm cell of early developmental process after pollination, vesicles that contain storage materials produced in rough endoplasmic reticulum incorporated into central vacuole. The central vacuole is gradually subdivided into several small-sized vacuoles and increased in number. Amorphous proteinaceous materials of high electron density are produced in rough endoplasmic reticulum. Rough endoplasmic reticulum increase in number and surround the protein body and vesicles circularly. Spherical proteinaceous granules with limited membrane appeared from the amorphous granules at the peripheral region of the rough endoplasmic reticulum. Gradually, storage materials are accumulated within the vacuole surrounded by spherosomes. Protein bodies are formed by interfusing between vacuoles and vesicles derived from rough endoplasmic reticulum which contained the amorphous protein of high electron density.

• BMB Reports
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• 제52권6호
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• pp.349-359
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• 2019

After the first research declaring the generation of human induced pluripotent stem cells (hiPSCs) in 2007, several attempts have been made to model neurodegenerative disease in vitro during the past decade. Parkinson's disease (PD) is the second most common neurodegenerative disorder, which is mainly characterized by motor dysfunction. The formation of unique and filamentous inclusion bodies called Lewy bodies (LBs) is the hallmark of both PD and dementia with LBs. The key pathology in PD is generally considered to be the alpha-synuclein (${\alpha}$-syn) accumulation, although it is still controversial whether this protein aggregation is a cause or consequence of neurodegeneration. In the present work, the recently published researches which recapitulated the ${\alpha}$-syn aggregation phenomena in sporadic and familial PD hiPSC models were reviewed. Furthermore, the advantages and potentials of using patient-derived PD hiPSC with focus on ${\alpha}$-syn aggregation have been discussed.

• 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.

• Biotechnology and Bioprocess Engineering:BBE
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• 제6권4호
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• pp.237-243
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• 2001

Substantial progress has been made towards understanding the folding mechanisms of proteins in virto and in vivo even though the general rules governing such folding events remain unknown. This paper reviews current folding models along with experimental approaches used to elucidate the folding pathways. Protein misfolding is discussed in relation to disease states, such as amyloidosis, and the recent findings on the mechanism of converting normally soluble proteins into amyloid fibrils through the formation of intermediates provide an insight into understanding the pathogenesis of amyloid formation and possible cules for the development of therapeutic treatments. Finally, some commonly adopted refolding strategies developed over the part decade are summarized.

• Biotechnology and Bioprocess Engineering:BBE
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• 제10권6호
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• pp.500-504
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• 2005

Fusion ferritin (heavy chain ferritin, $F_H+$ light chain ferritin, $F_L$), an iron-binding protein, was primarily purified from recombinant Escherichia coli by two-step sonications with urea [1]. Unfolded ferritin was refolded by gel filtration chromatography (GFC) with refolding enhancer, where 50 mM Na-phosphate (pH 7.4) buffer containing additives such as Tween 20, PEG, and L-arginine was used. Ferritin is a multimeric protein that contains approximately 20 monomeric units for full activity. Fusion ferritin was expressed in the form of inclusion bodies (IBs). The IBs were initially solubilized in 4 M urea denaturant. The refolding process was then performed by decreasing the urea concentration on the GFC column to form protein multimers. The combination of the buffer-exchange effect of GFC and the refolding enhancers in refolding buffer resulted in an efficient route for producing properly folded fusion ferritin.

• Bulletin of the Korean Chemical Society
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• 제31권9호
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• pp.2607-2612
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• 2010

Human tissue-type plasminogen activator (tPA) is a valuable thrombolytic agent used to successfully treat acute myocardial infarction, thromboembolic stroke, peripheral arterial occlusion, and venous thromboembolism. Recombinant tPA is accumulated as an inactive form in inclusion bodies of E. coli and is refolded in vitro, which is accompanied by extensive aggregation. In the present study, a tPA protease domain was expressed in an active soluble form in the cytosol of E. coli Rosetta-gami cells, which allowed disulfide bond formation and supplied the tRNA molecules required for six rarely used codons in E. coli. This strategy increased the amount of soluble protease domain protein and avoided the cumbersome refolding process. The purified protease domain not only degraded tPA substrate peptides but also formed a covalently bound complex with plasminogen activator inhibitor-1, as does full-length tPA. Soluble expression and purification of tPA domains may aid in functional analyses of this multi-domain protein, which has been implicated in many physiological and pathological processes.

• Journal of Microbiology and Biotechnology
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• 제19권4호
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• pp.362-367
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• 2009

In recombinant strains, many proteins and enzymes are expressed as inactive and insoluble inclusion bodies. For soluble expression of an active form of StyB, an NADH-flavin oxidoreductase, several recombinant Escherichia coli strains were developed and tested. Among them, strain BL21(DE3)pLysS effectively produced an active and soluble form of StyB as about 9% of the total protein content, when cultivated at $20^{\circ}C$ with 0.5 mM IPTG. The solubly expressed StyB has the highest oxidoreductase activity at pH 6.5-7.5 and $37^{\circ}C$. Substrate dependence profiles of the StyB-catalyzed reaction showed that the maximum specific activity($V_m$) and half saturation constant($K_m$) were $1,867{\pm}148\;U/mg$ protein and $51.6{\pm}11{\mu}M$ for NADH, and $1,274{\pm}34\;U/mg$ protein and $8.2{\pm}1.2{\mu}M$ for FAD, respectively. This indicates that solubly produced StyB has 6- to 9-fold higher oxidoreductase activities than the in vitro refolded StyB from inclusion bodies.

• Asian Pacific Journal of Cancer Prevention
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• 제14권12호
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• pp.7523-7527
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• 2013

Thymidylate synthase (TS) catalyzes the transfer of a methyl group from methylenetetrahydrofolate to dUMP to form dTMP. It is a primary target in the chemotherapy of colorectal cancers and some other neoplasms. In order to obtain pure protein for analysis of structure and biological function, an expression vector TS-pET28b (+) was constructed by inserting wild-type human thymidylate synthase (hTS) cDNA into pET28b (+). Then an expression strain was selected after transformation of the recombined plasmid into Rosetta (DE3). Fusion protein with His-tag was efficiently expressed in the form of inclusion bodies after IPTG induction and the content was approximately 40.0% of total bacteria proteins after optimizing expression conditions. When inclusion bodies were washed, dissolved and purified by Ni-NTA under denatured conditions, the purity was up to 90%. On SDS-PAGE and West-blotting, the protein band was found to match well with the predicted relative molecular mass-36kDa. Bioactivity was 0.1 U/mg. The results indicated that high-level expression of wild-type hTS cDNA can be achieved in prokaryotes with our novel method, facilitating research into related chemotherapy.