• 제목/요약/키워드: His-tagging

검색결과 9건 처리시간 0.021초

Effects of N-/C-Terminal Extra Tags on the Optimal Reaction Conditions, Activity, and Quaternary Structure of Bacillus thuringiensis Glucose 1-Dehydrogenase

  • Hyun, Jeongwoo;Abigail, Maria;Choo, Jin Woo;Ryu, Jin;Kim, Hyung Kwoun
    • Journal of Microbiology and Biotechnology
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    • 제26권10호
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    • pp.1708-1716
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    • 2016
  • Glucose dehydrogenase (GDH) is an oxidoreductase enzyme and is used as a biocatalyst to regenerate NAD(P)H in reductase-mediated chiral synthesis reactions. In this study, the glucose 1-dehydrogenase B gene (gdhB) was cloned from Bacillus thuringiensis subsp. kurstaki, and wild-type (GDH-BTWT) and His-tagged (GDH-BTN-His, GDH-BTC-His) enzymes were produced in Escherichia coli BL21 (DE3). All enzymes were produced in the soluble forms from E. coli. GDH-BTWT and GDH-BTN-His showed high specific enzymatic activities of 6.6 U/mg and 5.5 U/mg, respectively, whereas GDH-BTC-His showed a very low specific enzymatic activity of 0.020 U/mg. These results suggest that the intact C-terminal carboxyl group is important for GDH-BT activity. GDH-BTWT was stable up to 65℃, whereas GDH-BTN-His and GDH-BTC-His were stable up to 45℃. Gel permeation chromatography showed that GDH-BTWT is a dimer, whereas GDH-BTN-His and GDH-BTC-His are monomeric. These results suggest that the intact N- and C-termini are required for GDH-BT to maintain thermostability and to form its dimer structure. The homology model of the GDH-BTWT single subunit was constructed based on the crystal structure of Bacillus megaterium GDH (PDB ID 3AY6), showing that GDH-BTWT has a Rossmann fold structure with its N- and C-termini located on the subunit surface, which suggests that His-tagging affected the native dimer structure. GDH-BTWT and GDH-BTN-His regenerated NADPH in a yeast reductase-mediated chiral synthesis reaction, suggesting that these enzymes can be used as catalysts in fine-chemical and pharmaceutical industries.

One-step Purification of Poly-His Tagged Penicillin G Acylase Expressed in E. coli

  • Kim, Jin-Hee;Kang, Hye-Jin;Kim, Eung-Soo;Kim, Jeong-Ho;Koo, Yoon-Mo
    • Journal of Microbiology and Biotechnology
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    • 제14권2호
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    • pp.231-236
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    • 2004
  • The inexpensive large-scale production of pure PGA (Penicillin G Acylase) has been a commercial goal. PGA has been used as a model enzyme in the development of simple one-step purification methods. In this study, the purification of poly-His tagged PGA protein secreted into the periplasmic space was carried out by using immobilized metal-ion affinity chromatography (IMAC). The PGA gene was obtained from E. coli ATCC 11105. Codons encoding histidines were fused at the C-terminus of the PGA gene by PCR. E. coli JM109 harboring pPGA-HIS6 vector produced active his-tagged acylases in the presence of lac promoter during cultivation at $26^{\circ}C$. The maximum specific activity of the acylase purified by using one-step chromatography after osmotic shock was 38.5 U/mg and was recovered with the yield of 70%. Both 23 kDa ($\alpha$) and 62 kDa ($\beta$) subunits were recovered by using IMAC with just C-terminus tagging of the $\beta$ subunit. The purification of the periplasmic fraction by osmotic shock and that of purified acylase was increased by 2.6-fold and 19-fold, respectively, compared to the crude extract.

대장균에서 Chlamydia psittaci MOMP 유전자의 과발현과 순수분리 (Over-expression of Chlamydia psittaci MOMP in Escherichia coli and its purification)

  • 하정순;이도부;한상훈;임윤규;윤병수
    • 대한수의학회지
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    • 제46권1호
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    • pp.13-19
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    • 2006
  • Generally known psittacosis or ornithosis is a disease of birds caused by the bacterium Chlamydia psittaci. Humans are accidential hosts and are most commonly infected from avian sources. It raises hepatitis or neurosis. As major outer membrane protein (MOMP) of Chlamydia psittaci has been known to play a role in the avoidance of host immune defenses, research on developing a Chlamydia vaccine has focused on the MOMP. In this study, the gene encoding the major outer membrane protein (MOMP) of the Chlamydia psittaci strain 6BC was cloned and expressed in Escherichia coli strain M-15. The recombinant DNA was cloned by fusion prokaryotic expression vector pQE30-GFPII. Expression of the recombinant protein was performed in E. coli and was induced by IPTG. The size of expressed recombinant protein is 74.220 kDa (MOMP, 43.260 kDa; GFP expression region, 30 kDa; $6{\times}His$ tag, 960Da). This protein was purified by using his-tagging-inclusion body. Recombinant protein was reconfirmed through ELISA test and western blot with antibody against pQE30-GFPII. It will be useful antibody development.

교차결합 실험을 통한 루신 대응 조절 단백질의 4차 구조 분석 (Analysis of quaternary structure of leucine-responsive regulatory protein (Lrp) by crosslink experiments)

  • 이의호;로버트 포쿠;로이 트엔 응구엔;이찬용
    • 미생물학회지
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    • 제53권4호
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    • pp.297-303
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    • 2017
  • 루신-대응 조절 단백질(Lrp)은 18.8 kDa의 분자량을 갖는 글로벌 조절 단백질로서 대장균과 같은 장내세균과에서 많은 대사작용 오페론의 기능적 활성도를 조절한다. 단백질의 4차 구조를 규명하기 위한 목적으로 Lrp단백질 코드하는 유전자가 삽입된 재조합 플라스미드 pQE vector를 발현시킨 6 ${\times}$ His-tag Lrp 야생형과 $^3H$로 표지된 Lrp를 분리 정제한 후 cross linker들인 glutaraldehyde, 1,2,3,4-diepoxy-butane (DEB), ethylene glycol bis (succinimidyl succinate) (EGS)으로 cross link 실험을 수행하여 Lrp가 $0.3{\mu}M$ 이하의 낮은 농도에서나 $5{\mu}M$의 높은 농도에서 이량체, 사량체, 육량체, 팔량체로 존재할 수 있음을 확인하였다.

Bi-functional Activities of Chimeric Lysozymes Constructed by Domain Swapping between Bacteriophage T7 and K11 Lysozymes

  • Alcantara, Ethel H.;Kim, Dong-Hee;Do, Su-Il;Lee, Sang-Soo
    • BMB Reports
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    • 제40권4호
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    • pp.539-546
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    • 2007
  • The lysozymes encoded by bacteriophage T7 and K11 are both bifunctional enzymes sharing an extensive sequence homology (75%). The constructions of chimeric lysozymes were carried out by swapping the N-terminal and C-terminal domains between phage T7 and K11 lysozymes. This technique generated two chimeras, T7K11-lysozyme (N-terminal T7 domain and C-terminal K11 domain) and K11T7-lysozyme (N-terminal K11 domain and C-terminal T7 domain), which are both enzymatically active. The amidase activity of T7K11-lysozyme is comparable with the parental enzymes while K11T7-lysozyme exhibits an activity that is approximately 45% greater than the wild-type lysozymes. Moreover, these chimeric constructs have optimum pH of 7.2-7.4 similar to the parental lysozymes but exhibit greater thermal stabilities. On the other hand, the chimeras inhibit transcription comparable with the parental lysozymes depending on the source of their N-terminals. Taken together, our results indicated that domain swapping technique localizes the N-terminal region as the domain responsible for the transcription inhibition specificity of the wild type T7 and K11 lysozymes. Furthermore, we were able to develop a simple and rapid purification scheme in purifying both the wild-type and chimeric lysozymes.

발광세균 Photobacterium leiognathi의 돌연변이 아미노-말단 루마진 단백질들의 제조, 발현 및 정제 (Construction, Expression, and Purification of N-Terminal Variants of Lumazine Protein from Photobacterium leiognathi)

  • 강경숙;김소영;최지선;김영두;로버트 포쿠;남기석;이찬용
    • 미생물학회지
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    • 제49권2호
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    • pp.205-210
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    • 2013
  • 루마진 단백질은 발광 세균인 Photobacterium 종에서 추출된 형광성 단백질이다. 형광성을 지닌 최소 크기의 Photobacterium leiognathi 야생형 아미노-말단 도메인 루마진 단백질(N-terminal domain of lumazine protein 118 wt)과 여러 영역에 tryptophan을 생성시킨 돌연변이 단백질들(N-LumP 118 V41W, S48W, T50W, D64W, A66W)을 코드하는 유전자들을 위치 지정 돌연변이(Site Directed Mutagenesis)와 중합효소 연쇄 반응(Polymerase Chain Reaction)을 통해 제조하였다. 위의 유전자들이 포함된 재조합 플라스미드를 대장균에 형질 전환시켜 과발현시키는 최적의 조건을 찾았으며, 발현된 야생형 및 돌연변이 아미노-말단 영역 루마진 단백질을 6X-His tag system을 이용하여 정제 하였다. 흡광 및 형광 분광광도계를 이용한 실험 결과 이들 단백질들은 리간드인 6,7-dimethyl-8-ribityllumazine과 결합하여 형광성을 보유함을 보였다. 따라서 이들은 형광성을 지니게 되는 최소 크기의 루마진 단백질일 뿐만 아니라 형광성을 지닌 아미노산인 tryptophan이 여러 위치에 유일하게 존재함으로써 배향성 및 거리 등의 단백질의 구조 및 결합에 관한 심도 있는 연구에 탐침자로써 유용하게 활용 될 수 있을 것이다.

폭소노미 사이트를 위한 랭킹 프레임워크 설계: 시맨틱 그래프기반 접근 (A Folksonomy Ranking Framework: A Semantic Graph-based Approach)

  • 박현정;노상규
    • Asia pacific journal of information systems
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    • 제21권2호
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    • pp.89-116
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    • 2011
  • In collaborative tagging systems such as Delicious.com and Flickr.com, users assign keywords or tags to their uploaded resources, such as bookmarks and pictures, for their future use or sharing purposes. The collection of resources and tags generated by a user is called a personomy, and the collection of all personomies constitutes the folksonomy. The most significant need of the folksonomy users Is to efficiently find useful resources or experts on specific topics. An excellent ranking algorithm would assign higher ranking to more useful resources or experts. What resources are considered useful In a folksonomic system? Does a standard superior to frequency or freshness exist? The resource recommended by more users with mere expertise should be worthy of attention. This ranking paradigm can be implemented through a graph-based ranking algorithm. Two well-known representatives of such a paradigm are Page Rank by Google and HITS(Hypertext Induced Topic Selection) by Kleinberg. Both Page Rank and HITS assign a higher evaluation score to pages linked to more higher-scored pages. HITS differs from PageRank in that it utilizes two kinds of scores: authority and hub scores. The ranking objects of these pages are limited to Web pages, whereas the ranking objects of a folksonomic system are somewhat heterogeneous(i.e., users, resources, and tags). Therefore, uniform application of the voting notion of PageRank and HITS based on the links to a folksonomy would be unreasonable, In a folksonomic system, each link corresponding to a property can have an opposite direction, depending on whether the property is an active or a passive voice. The current research stems from the Idea that a graph-based ranking algorithm could be applied to the folksonomic system using the concept of mutual Interactions between entitles, rather than the voting notion of PageRank or HITS. The concept of mutual interactions, proposed for ranking the Semantic Web resources, enables the calculation of importance scores of various resources unaffected by link directions. The weights of a property representing the mutual interaction between classes are assigned depending on the relative significance of the property to the resource importance of each class. This class-oriented approach is based on the fact that, in the Semantic Web, there are many heterogeneous classes; thus, applying a different appraisal standard for each class is more reasonable. This is similar to the evaluation method of humans, where different items are assigned specific weights, which are then summed up to determine the weighted average. We can check for missing properties more easily with this approach than with other predicate-oriented approaches. A user of a tagging system usually assigns more than one tags to the same resource, and there can be more than one tags with the same subjectivity and objectivity. In the case that many users assign similar tags to the same resource, grading the users differently depending on the assignment order becomes necessary. This idea comes from the studies in psychology wherein expertise involves the ability to select the most relevant information for achieving a goal. An expert should be someone who not only has a large collection of documents annotated with a particular tag, but also tends to add documents of high quality to his/her collections. Such documents are identified by the number, as well as the expertise, of users who have the same documents in their collections. In other words, there is a relationship of mutual reinforcement between the expertise of a user and the quality of a document. In addition, there is a need to rank entities related more closely to a certain entity. Considering the property of social media that ensures the popularity of a topic is temporary, recent data should have more weight than old data. We propose a comprehensive folksonomy ranking framework in which all these considerations are dealt with and that can be easily customized to each folksonomy site for ranking purposes. To examine the validity of our ranking algorithm and show the mechanism of adjusting property, time, and expertise weights, we first use a dataset designed for analyzing the effect of each ranking factor independently. We then show the ranking results of a real folksonomy site, with the ranking factors combined. Because the ground truth of a given dataset is not known when it comes to ranking, we inject simulated data whose ranking results can be predicted into the real dataset and compare the ranking results of our algorithm with that of a previous HITS-based algorithm. Our semantic ranking algorithm based on the concept of mutual interaction seems to be preferable to the HITS-based algorithm as a flexible folksonomy ranking framework. Some concrete points of difference are as follows. First, with the time concept applied to the property weights, our algorithm shows superior performance in lowering the scores of older data and raising the scores of newer data. Second, applying the time concept to the expertise weights, as well as to the property weights, our algorithm controls the conflicting influence of expertise weights and enhances overall consistency of time-valued ranking. The expertise weights of the previous study can act as an obstacle to the time-valued ranking because the number of followers increases as time goes on. Third, many new properties and classes can be included in our framework. The previous HITS-based algorithm, based on the voting notion, loses ground in the situation where the domain consists of more than two classes, or where other important properties, such as "sent through twitter" or "registered as a friend," are added to the domain. Forth, there is a big difference in the calculation time and memory use between the two kinds of algorithms. While the matrix multiplication of two matrices, has to be executed twice for the previous HITS-based algorithm, this is unnecessary with our algorithm. In our ranking framework, various folksonomy ranking policies can be expressed with the ranking factors combined and our approach can work, even if the folksonomy site is not implemented with Semantic Web languages. Above all, the time weight proposed in this paper will be applicable to various domains, including social media, where time value is considered important.

아미노-말단 리보플라빈 생성효소 단백질의 형광 특성 (Spectrofluorometric Characteristics of the N-Terminal Domain of Riboflavin Synthase)

  • 김류련;이정환;남기석;고경원;이찬용
    • 미생물학회지
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    • 제47권1호
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    • pp.14-21
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    • 2011
  • 리보플라빈 생성효소(riboflavin synthase)는 기질인 두 분자의 6,7-dimetyl-8-ribityllumazine과 결합 후, 4-탄소 단위(4-carbon unit)의 자리 옮김 반응을 거쳐 한 분자의 리보플라빈과 한 분자의 pyrimidine 유도체를 형성하는 반응을 촉매한다. 대장균(Escherichia coli) 리보플라빈 생성효소의 아미노-말단 도메인 절반(N-terminal domain half)과 카복시-말단 도메인 절반(C-terminal domain half)은 매우 유사한 내부 자체 아미노산 서열(intra-molecular amino acid sequence)을 갖는다. 아미노-말단 영역 리보플라빈 생성효소(N-RS) 단백질의 구조와 형광 특성을 알아보기 위하여 중합효소 연쇄 반응과 위치지정 돌연변이를 통하여 10개 이상의 돌연변이 아미노-말단 리보플라빈 생성효소 단백질을 코드 하는 유전자를 증폭시켜 pQE30 벡터에 삽입한 재조합 플라스미드를 제조하여, 과발현시킨 후 분리 정제하였다. 대부분의 아미노-말단 도메인 리보플라빈 생성효소의 돌연변이 단백질들은 야생형과 같이 형광성 리간드인 6,7-dimetyl-8-ribityllumazine 혹은 리보플라빈과 결합할 수 있는 능력을 지니고 있었으나, N-RS C47D, N-RS ET66,67DQ 돌연변이 단백질의 경우는 리간드와의 결합능력이 현저히 떨어져 형광을 띠지 않았다. 대부분의 돌연변이 단백질들의 형광 세기는 야생형 단백질(N-RS wt)보다 낮았으나, N-RS C48S는 예외적으로 야생형 단백질에 비해 2배 이상의 형광세기를 가졌다. 이와 같은 결과를 바탕으로 리보플라빈 생성효소와 형광성 리간드 사이의 상호작용을 예측 할 수 있으며, N-RS C48S 돌연변이 단백질의 형광성을 활용하여 효과적으로 효소 저해제를 발굴할 수 있는 고속다중 스크리닝 법(high-throughput screening system)으로써 활용될 수 있을 것이다.

전자 팔찌를 이용한 전자 출석부.어린이 보호 장치 시스템 (Electronic Roll Book using Electronic Bracelet.Child Safe-Guarding Device System)

  • 문승진;김태남;김판수
    • 지능정보연구
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    • 제17권4호
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    • pp.143-155
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    • 2011
  • 본 논문에서는 RFID 기반의 전자 팔찌를 이용한 전자 출석부 및 어린이 보호팔찌 시스템을 제안한다. 제안된 시스템에서는 임베디드시스템 기반의 RFID 기술을 응용한 전자 출석부가 필요하며, 위치 추적과 동시에 범인의 전자 팔찌와 반응할 수 있는 GPS 시스템이 있어야 한다. 또한 이동 통신 모듈을 이용한 RFID 전자 팔찌와 임베디드시스템 기반의 RFID 출입 통제 시스템이 필요하며 상황정보를 전송 받을 수 있는 스마트폰 등의 모바일 시스템이 있어야 한다. 이렇게 동시에 연동될 수 있는 장비와 시스템들을 융합하여 사용한다면 아이들이 안전하게 유치원이나 학교를 다닐 수 있을 것이다. 본 논문의 구성은 RFID 리더기와 임베디드시스템 기반의 전자 출석부(고정용 리더기) 및 출입 통제 시스템, RFID 태그와 리더기, GPS 및 이동 통신 모듈(CELL 방식)를 활용한 추적 시스템을 가진 전자 팔찌로 이루어져 있다.