• Journal of Microbiology and Biotechnology
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• 제31권4호
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• pp.570-583
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• 2021

Pyrococcus furiosus α-amylase can hydrolyze α-1,4 linkages in starch and related carbohydrates under hyperthermophilic condition (~ 100℃), showing great potential in a wide range of industrial applications, while its relatively low productivity from heterologous hosts has limited the industrial applications. Bacillus subtilis, a gram-positive bacterium, has been widely used in industrial production for its non-pathogenic and powerful secretory characteristics. This study was conducted to increase production of P. furiosus α-amylase in B. subtilis through three strategies. Initial experiments showed that co-expression of P. furiosus molecular chaperone peptidyl-prolyl cis-trans isomerase through genomic integration mode, using a CRISPR/Cas9 system, increased soluble amylase production. Therefore, considering that native P. furiosus α-amylase is produced within a hyperthermophilic environment and is highly thermostable, heat treatment of intact culture at 90℃ for 15 min was performed, thereby greatly increasing soluble amylase production. After optimization of the culture conditions (nitrogen source, carbon source, metal ion, temperature and pH), experiments in a 3-L fermenter yielded a soluble activity of 3,806.7 U/ml, which was 3.3- and 28.2-fold those of a control without heat treatment (1,155.1 U/ml) and an empty expression vector control (135.1 U/ml), respectively. This represents the highest P. furiosus α-amylase production reported to date and should promote innovation in the starch liquefaction process and related industrial productions. Meanwhile, heat treatment, which may promote folding of aggregated P. furiosus α-amylase into a soluble, active form through the transfer of kinetic energy, may be of general benefit when producing proteins from thermophilic archaea.

• Asian Pacific Journal of Cancer Prevention
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• 제14권4호
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• pp.2383-2386
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• 2013

Senescence marker protein 30 (SMP30), a hepatocellular carcinoma (HCC) associated antigen, was earlier shown by our research group to be highly expressed in HCC paracancerous tissues, but have low levels in HCC tissues. In order to detect anti-SMP30 antibody in serum of HCC patients, we established pET30a-SMP30 and pColdIII-SMP30 expression systems in Escherichia coli. However, the expression product was mainly in the form of inclusion bodies. In this research, we used several combinations of chaperones, four molecular chaperone plasmids with pET30a-SMP30 and five molecular chaperone plasmids with pColdIII-SMP30 to increase the amount of soluble protein. Results showed that co-expression of HIS-SMP30 with pTf16, combined with the addition of osmosis-regulator, and a two-step expression resulted in the highest enhancement of solubility. A total of 175 cases of HCC serum were studied by ELISA to detect anti-SMP30 antibody with recombinant SMP30 protein. Some 22 were positive and x2 two-sided tests all showed P>0.05, although it remained unclear whether there was a relationship between positive cases and clinical diagnostic data.

• 한국산학기술학회논문지
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• 제21권9호
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• pp.559-568
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• 2020

세포 분열 및 성장 촉진에 영향을 주는 상피세포 성장인자(Epidermal Growth Factor, EGF)는 다양한 의학적 용도를 갖고 있는 호르몬 단백질이다. 본 연구에서는 human EGF 유전자를 대장균 코돈에 최적화 하고 pRSET 벡터에 클로닝하여 발현벡터를 구축하였다. Human EGF를 봉입체가 아닌 활성이 있는 형태로의 과량 발현을 위해 코돈의 최적화와 더불어 최초로 샤페론 공발현이 시도되었다. 발현된 Native protein 형태의 재조합 human EGF는 고순도로 정제하기 위해 Ion Exchange Chromatography를 2번 연속적으로 수행하여 순수 분리 정제되었고, ELISA 분석결과 99% 이상으로 재조합 EGF의 활성도가 상업용 EGF와 유사하게 나타났으며, 세포증식시험 결과 인간 재조합 EGF는 인체 피부 섬유아세포의 세포증식을 촉진하는 것으로 확인 되었다. 본 연구의 인간 EGF 발현 시스템은 양적인 측면 뿐 아니라 성공적인 활성형태의 발현으로 추가적인 재접힘 과정 및 N 말단의 융합부분을 제거하기 위한 크로마토그래피 작업이 필요가 없다는 점에서 기존의 방법들에 대체 될 수 있는 효과적인 인간 EGF 발현 시스템을 제공하고 있다.

• Animal cells and systems
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• 제14권1호
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• pp.1-10
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• 2010

Previously we showed that CHIP, a co-chaperone of Hsp70 and E3 ubiquitin ligase, can promote the degradation of mutant SOD1 linked to familial amyotrophic lateral sclerosis (fALS) via a mechanism not involving SOD1 ubiquitylation. Here we present evidence that CHIP functions in the interaction of mutant SOD1 with 26S proteasomes. Bag-1, a coupling factor between molecular chaperones and the proteasomes, formed a complex with SOD1 in an hsp70-dependent manner but had no direct effect on the degradation of mutant SOD1. Instead, Bag-1 stimulated interaction between CHIP and the proteasome-associated protein VCP (p97), which do not associate normally. Over-expressed CHIP interfered with the association between mutant SOD1 and VCP. Conversely, the binding of CHIP to mutant SOD1 was inhibited by VCP, implying that the chaperone complex and proteolytic machinery are competing for the common substrates. Finally we observed that mutant SOD1 strongly associated with the 19S complex of proteasomes and CHIP over-expression specifically reduced the interaction between S6/S6' ATPase subunits and mutant SOD1. These results suggest that CHIP, together with ubiquitin-binding proteins such as Bag-1 and VCP, promotes the degradation of mutant SOD1 by facilitating its translocation from ATPase subunits of 19S complex to the 20S core particle.

• Toxicological Research
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• 제24권2호
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• pp.129-135
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• 2008

The results of recent studies indicate that high levels of free fatty acids(FFAs) and adipokines may be the main causes of non-alcoholic liver disease; however, the molecular mechanism that links FFAs to lipotoxicity remains unclear. In the present study, we treated HepG2 cells with FFA(either palmitate or oleate) to investigate the mechanisms involved in lipotoxicity in the liver cells. We also treated cells with palmitate in the presence of a chemical chaperone, 4-phenylbutyric acid(PBA), to confirm the involvement of ER stress in lipotoxicity. Palmitate significantly induced cytotoxicity in dose- and time-dependent manners. Apoptosis was also significantly induced by palmitate as measured by caspase-3 activity and DAPI staining. Palmitate led to increased expressions of the spliced form of X-box-protein(Xbp)-1 mRNA and C/EBP homologous transcription factor(CHOP) protein, suggesting activation of the unfolded-protein response. PBA co-incubation significantly attenuated apoptosis induced by palmitate. The above data demonstrate that high levels of palmitate induce apoptosis via the mediation of ER stress in the liver cells and that chemical chaperones act to modulate ER stress and accompanying apoptosis.

• Journal of Microbiology and Biotechnology
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• 제25권2호
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• pp.247-254
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• 2015

In this presentation, I describe the expression and purification of the recombinant liver X receptor β-ligand binding domain proteins in E. coli using a commercially available double cistronic vector, pACYCDuet-1, to express the receptor heterodimer in a single cell as the soluble form. I describe here the expression and characterization of a biologically active heterodimer composed of the liver X receptor β-ligand binding domain and retinoid X receptor α-ligand binding domain. Although many of these proteins were previously seen to be produced in E. coli as insoluble aggregates or "inclusion bodies", I show here that as a form of heterodimer they can be made in soluble forms that are biologically active. This suggests that co-expression of the liver X receptor β-ligand binding domain with its binding partner improves the solubility of the complex and probably assists in their correct folding, thereby functioning as a type of molecular chaperone.

• 한국미생물·생명공학회지
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• 제42권2호
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• pp.190-193
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• 2014

CMP-Neu5Ac synthetase는 sialyated 된 glycoconjugates의 전구체로 사용되는 CMP-Neu5Ac를 합성하는데 관여하는 주요 효소이다. Escherichia coli K1에서 유래한 CMP-Neu5Ac synthetase 유전자 (neuA)는 평소 E. coli BL21(DE3)에서 비수용성으로 생성되는데, 이를 수용성 단백질로 생산하고자 여러 가지 샤페론 단백질 동시 발현기술을 이용하였다. 이를 위해, GroEL-ES와 DnaK-DnaJ-GrpE를 암호화하는 pG-KJE8 plasmid와 neuA를 동시 형질전환 시켰고 0.01 mM IPTG와 0.005 mg/ml의 L-arabinose로 유도하여 $20^{\circ}C$에서 발현시켰다. 그 결과, E. coli에서의 수용성 CMP-Neu5Ac Synthetase 생산이 현저하게 증가하였다.

• Journal of Microbiology and Biotechnology
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• 제29권6호
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• pp.944-951
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• 2019

Lipases are industrial enzymes that catalyze both triglyceride hydrolysis and ester synthesis. The overexpression of lipase genes is considered one of the best approaches to increase the enzymatic production for industrial applications. Subfamily I.2. lipases require a chaperone or foldase in order to become a fully-activated enzyme. The goal of this research was to isolate, clone, and co-express genes that encode lipase and foldase from Burkholderia territorii GP3, a lipolytic bacterial isolate obtained from Mount Papandayan soil via growth on Soil Extract Rhodamine Agar. Genes that encode for lipase (lipBT) and foldase (lifBT) were successfully cloned from this isolate and co-expressed in the E. coli BL21 background. The highest expression was shown in E. coli BL21 (DE3) pLysS, using pET15b expression vector. LipBT was particulary unique as it showed highest activity with optimum temperature of $80^{\circ}C$ at pH 11.0. The optimum substrate for enzyme activity was $C_{10}$, which is highly stable in methanol solvent. The enzyme was strongly activated by $Ca^{2+}$, $Mg^{2+}$, and strongly inhibited by $Fe^{2+}$ and $Zn^{2+}$. In addition, the enzyme was stable and compatible in non-ionic surfactant, and was strongly incompatible in ionic surfactant.

• Molecules and Cells
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• 제20권1호
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• pp.1-16
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• 2005

The peptidyl transferase center (PTC) is located in a protein free environment, thus confirming that the ribosome is a ribozyme. This arched void has dimensions suitable for accommodating the 3'ends of the A-and the P-site tRNAs, and is situated within a universal sizable symmetry-related region that connects all ribosomal functional centers involved in amino-acid polymerization. The linkage between the elaborate PTC architecture and the A-site tRNA position revealed that the A-to P-site passage of the tRNA 3'end is performed by a rotatory motion, which leads to stereochemistry suitable for peptide bond formation and for substrate mediated catalysis, thus suggesting that the PTC evolved by genefusion. Adjacent to the PTC is the entrance of the protein exit tunnel, shown to play active roles in sequence-specific gating of nascent chains and in responding to cellular signals. This tunnel also provides a site that may be exploited for local co-translational folding and seems to assist in nascent chain trafficking into the hydrophobic space formed by the first bacterial chaperone, the trigger factor. Many antibiotics target ribosomes. Although the ribosome is highly conserved, subtle sequence and/or conformational variations enable drug selectivity, thus facilitating clinical usage. Comparisons of high-resolution structures of complexes of antibiotics bound to ribosomes from eubacteria resembling pathogens, to an archaeon that shares properties with eukaryotes and to its mutant that allows antibiotics binding, demonstrated the unambiguous difference between mere binding and therapeutical effectiveness. The observed variability in antibiotics inhibitory modes, accompanied by the elucidation of the structural basis to antibiotics mechanism justifies expectations for structural based improved properties of existing compounds as well as for the development of novel drugs.

• 약학회지
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• 제50권4호
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• pp.263-267
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• 2006

CD1d is an unique antigen presenting molecule which provides antigenic repertoires to NKT cells. To examine molecules required for CD1d antigen presentation, we determined an interaction between CD1d and several endoplasmic reticulum (ER) resident molecular chaperones by co-immunoprecipitation. Results indicated that calnexin and calreticulin seem to be bound to mouse CD1d, but TAP and tapasin do not bind. Further, we screened an yeat two hybrid system to identify proteins that help mouse CD1d transportation in the cytosol. We found that two proteins, heat shock protein a sub-unit $(Hsp90{\alpha})$ and protein kinase C and casein kinase substrate in neurons 3 (PACSIN-3), interact with CD1d. Future study will be focus on the role of these molecules during the CD1d antigen presentation.