• 한국생물물리학회:학술대회논문집
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• 한국생물물리학회 1996년도 정기총회 및 학술발표회
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• pp.13-13
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• 1996

Aggregation of human $\alpha$$_1$-antitrypsin ($\alpha$$_1$-AT) during folding occurs both in vitro and in vivo. In vivo aggregates of mutant $\alpha$$_1$-AT such as $M_{malton}$ (Phe52 deleted) and Z (Glu342 longrightarrowLys) variants have pathological consequences. In order to analyze the process of $\alpha$$_1$-AT aggregation in detail, the folding-unfolding kinetics of $\alpha$$_1$-AT was examined by monitoring intrinsic Trp fluorescence and ANS binding. (omitted)

• Journal of Microbiology and Biotechnology
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• 제11권1호
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• pp.92-96
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• 2001

Effect of environmental factors on the expression of soluble forms of Bacillus macerans cyclodextrin glycosyltransferase in recombinant Escherichia coli BL21(DE3)pLysE:pTCGT1 were investigated. The amount of soluble CGTase produced in the cell was measured by determining its enzymatic activity. The soluble fractionof the enzyme was increased by lowering the culture temperature to $30{\circ}C$ and medium pH to 5.8 compared to the enzyme production in LB medium at $37^{\circ}C$ and pH7.0. Addition of 0.2 M NaCl enhanced enzyme expression levels at the expense of cell growth. Glycine betaine that was added after 3 h of induction protected not only the cell growth from hig osmotic pressue but also hepld in vivo folding of CGTase in recombinant E. coli. Addition of 1 mM $CaCl_2$ was also effective in the expression of soluble CGTase, resulting in 15 U/ml of the enzyme activity.

• 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.

• 생명과학회지
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• 제6권4호
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• pp.304-312
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• 1996

최근에 발표된 연구 결과에 의하면 대장균 트립토판 중합효소(tryptophan synthase) $\alpha$ 소단위체에서 33번 잔기의 세린과 112번 잔기의 아스팔산은 이 효소의 구조 형성 과정(folding)에 관여하는 것으로 보여진다. 이들 효소를 대장균내에서 과발현시켰을 때, SL33(잔기 33번의 세린이 류신으로 치환) 효소는 대부분 웅집된 덩어리형태의 효소로 생성되고, DG112(잔기 112번의 아스팔산이 글리신으로 치환) 효소와 DN112(아스랄산이 아스파라진으로 치환) 효소는 야생형 효소와 비슷한 양의 수용성형태로 생성된다고 보고된 바 있다. 본 연구에서는 이 효소의 구조 형성 과정동안 33번 잔기와 112번 잔기가 상호 작용하는 가를 조사하기 위하여 주 자리 잔기 치환 단백질을 만들어 이들의 성질을 단일 잔기 치환 단백질들과 비교하였다. 이들을 대장균내에서 젖당으로 과생산하여 전기영동으로 조사하여 본 결과 SL33 단일 잔기 치환 효소에 비해 SL33/DG112 이중 치환 효소는 현격히 보다 많은 양이 응집된 형태의 불용성 효소로 생성 되며, SL33/DN112효소는 많은 양의 효소들이 가용성 효소로 생성되었다. Densitometer로 정량한 결과 SL33/DN112 치환 소단위체는 SL33 치환 소단위체에 비해 가용성 형태의 양에 대한 응집된 형태의 양의 비가 6시간 젖당처리시 약 5배, 22시간 젖당처리시 약 13배 증가하였으며, SL33/DN112 치환 $\alpha$ 소단위체는 그 비가 1/4-1/3로 감소했다. 이러한 결과는 331번 위치에서 세린이 류신으로 치환되었을 때 나타나는 구조 형성 과정의 변화를 112번 위치에 새로 치환된 글리신 또는 아스파라진이 영향을 미쳐, 구조 형성 솨정 변화가 더욱 더 변화했거나 원상태로 어느 정도 회복되었음을 의미한다. 이 결과는 대장균 트립토판 중합효소 $\alpha$ 소단위체의 N 말단 도메인(N-terminal domain)의 구조 형성 과정시 33번 잔기와 112번 잔기가 상호작용을 하고 있음을 시사해 주고 있다.

• 생명과학회지
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• 제9권2호
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• pp.146-152
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• 1999

본 연구에서는 대장균 트립토판 중합효소(tryptophan synthase) $\alpha$ 소단위체의 폴딩에 있어 24번 잔기의 역할을 보고자 하였다. 24번 잔기인 트레오닌이 메티오닌, 알라닌, 세린, 류신 또는 리신으로 치환된 단백질를 대장균내에서 과량 발현시켜 수용성 구조의 양과 비수용성인 inclusion body양을 조사하였다. 그 결과 메티오닌이나 류신으로 치환된 $\alpha$ 소단위체는 트레오닌이 있는 소단위체와 마찬가지로 수용성 구조가 대부분을 차지하였다. 반면, 세린이나 알라닌 또는 리신으로 치환된 단백질들은 많은 양이 inclusion body로 발현되었다. 이 결과는 트립토판 중합효소 $\alpha$ 소단위체의 폴딩에 있어서 24번 잔기가 관여하고 있음을 제시하고 있다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XII)
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• pp.55-55
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• 2003

• BMB Reports
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• 제38권3호
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• pp.259-265
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• 2005

Proteins must fold into their correct three-dimensional conformation in order to attain their biological function. Conversely, protein aggregation and misfolding are primary contributors to many devastating human diseases, such as prion-mediated infections, Alzheimer's disease, type II diabetes and cystic fibrosis. While the native conformation of a polypeptide is encoded within its primary amino acid sequence and is sufficient for protein folding in vitro, the situation in vivo is more complex. Inside the cell, proteins are synthesized or folded continuously; a process that is greatly assisted by molecular chaperones. Molecular chaperones re a group of structurally diverse and mechanistically distinct proteins that either promote folding or prevent the aggregation of other proteins. With our increasing understanding of the proteome, it is becoming clear that the number of proteins that can be classified as molecular chaperones is increasing steadily. Many of these proteins have novel but essential cellular functions that differ from that of more 'conventional' chaperones, such as Hsp70 and the GroE system. This review focuses on the emerging role of molecular chaperones in protein quality control, i.e. the mechanism that rids the cell of misfolded or incompletely synthesized polypeptides that otherwise would interfere with normal cellular function.

• Journal of Microbiology and Biotechnology
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• 제26권2호
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• pp.337-346
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• 2016

Three combinations of molecular chaperones from Escherichia coli (i.e., DnaK-DnaJ-GrpE-GroEL-GroES, GroEL-GroES, and DnaK-DnaJ-GrpE) were overproduced in E. coli BL21, and their in vitro stabilizing effects on a nitrile hydratase (NHase) were assessed. The optimal gene combination, E. coli groEL-groES (ecgroEL-ES), was introduced into Rhodococcus ruber TH3. A novel engineered strain, R. ruber TH3G was constructed with the native NHase gene on its chromosome and the heterologous ecgroEL-ES genes in a shuttle plasmid. In R. ruber TH3G, NHase activity was enhanced 37.3% compared with the control, TH3. The in vivo stabilizing effect of ecGroEL-ES on the NHase was assessed using both acrylamide immersion and heat shock experiments. The inactivation behavior of the in vivo NHase after immersion in a solution of dynamically increased concentrations of acrylamide was particularly evident. When the acrylamide concentration was increased to 500 g/l (50%), the remaining NHase activity in TH3G was 38%, but in TH3, activity was reduced to 10%. Reactivation of the in vivo NHases after varying degrees of inactivation was further assessed. The activity of the reactivated NHase was more than 2-fold greater in TH3G than in TH3. The hydration synthesis of acrylamide catalyzed by the in vivo NHase was performed with continuous acrylonitrile feeding. The final concentration of acrylamide was 640 g/l when catalyzed by TH3G, compared with 490 g/l acrylamide by TH3. This study is the first to show that the chaperones ecGroEL-ES work well in Rhodococcus and simultaneously possess protein-folding assistance functions and the ability to stabilize and reactivate the native NHases.

• Journal of Microbiology and Biotechnology
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• 제27권3호
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• pp.644-647
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• 2017

Peptidyl prolyl cis/trans isomerases (PPIases) catalyze the cis/trans isomerization of peptidyl-prolyl peptide bonds preceding prolines. We investigated the protein-protein interaction between a 22 kDa PPIase (VaFKBP22, an FK506-binding protein) and the molecular chaperone DnaK derived from Vibrio anguillarum O1 (VaDnaK) using GST pull-down assays and a bacterial two-hybrid system for in vivo and in vitro studies, respectively. Furthermore, we analyzed the three-dimensional structure of the protein-protein interaction. Based on our results, VaFKBP22 appears to act as a cochaperone of VaDnaK, and contributes to protein folding and stabilization via its peptidyl-prolyl cis/trans isomerization activity.

• Genomics & Informatics
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• 제21권3호
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• pp.30.1-30.13
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• 2023

Ephs belong to the largest family of receptor tyrosine kinase and are highly conserved both sequentially and structurally. The structural organization of Eph is similar to other receptor tyrosine kinases; constituting the extracellular ligand binding domain, a fibronectin domain followed by intracellular juxtamembrane kinase, and SAM domain. Eph binds to respective ephrin ligand, through the ligand binding domain and forms a tetrameric complex to activate the kinase domain. Eph-ephrin regulates many downstream pathways that lead to physiological events such as cell migration, proliferation, and growth. Therefore, considering the importance of Eph-ephrin class of protein in tumorigenesis, 7,620 clinically reported missense mutations belonging to the class of variables of unknown significance were retrieved from cBioPortal and evaluated for pathogenicity. Thirty-two mutations predicted to be pathogenic using SIFT, Polyphen-2, PROVEAN, SNPs&GO, PMut, iSTABLE, and PremPS in-silico tools were found located either in critical functional regions or encompassing interactions at the binding interface of Eph-ephrin. However, seven were reported in nonsmall cell lung cancer (NSCLC). Considering the relevance of receptor tyrosine kinases and Eph in NSCLC, these seven mutations were assessed for change in the folding pattern using molecular dynamic simulation. Structural alterations, stability, flexibility, compactness, and solvent-exposed area was observed in EphA3 Trp790Cys, EphA7 Leu749Phe, EphB1 Gly685Cys, EphB4 Val748Ala, and Ephrin A2 Trp112Cys. Hence, it can be concluded that the evaluated mutations have potential to alter the folding pattern and thus can be further validated by in-vitro, structural and in-vivo studies for clinical management.