• BMB Reports
• /
• 제33권3호
• /
• pp.242-248
• /
• 2000

Phage display of proteins is a powerful tool for protein engineering since a vast library of sequences can be rapidly screened for a specific property. In this study, we develop da new phage display vector that was derived from a pET-25b(+) vector. The pET-25b(+) was modified in order that the expressed protein would have a T7-tag at the amino terminus and GpS (a major coat protein of M13 phage) at the carboxyl terminus. Another vector without the gp8 gene was also constructed. The newly developed phagemid vectors have several advantageous features. First, it is easy to examine whether or not the target proteins are functional and faithfully transported into the periplasmic space. This feature is due to the fact that recombinant proteins are produced abundantly in the pET system. Second, the T7-tag makes it possible to detect any target proteins that are displayed on the surface of filamentous bacteriophage. To verify the utility of the vector, the clones containing the glutathione S-transferase (GST) gene as a target were examined. The result showed that the GST produced from the recombinant vector was successfully transported into the periplasmic space and had the anticipated enzyme activity. Western blot analysis using a T7-tag antibody also showed the presence of the target protein displayed on the surface of the phage. The phages prepared from the recombinant clones were able to bind to glutathione-Sepharose and then eluted with glutathione. These results showed that the new vectors developed in this study are useful for the phage display of proteins.

• 한국정보과학회논문지:소프트웨어및응용
• /
• 제36권9호
• /
• pp.749-757
• /
• 2009

인터넷 사용이 대중화되면서 개인이 정보의 또는 검색할 주제에 따라 원하는 정보에 쉽게 접근할 수 있다. 이때 다양한 구조를 갖는 자료들의 속성을 잘 나타내는 메타데이터를 이용하면 검색의도에 보다 정확하게 부합하는 검색 결과를 얻을 수 있어 다양한 연구가 지속되고 있다. 본 연구는 소그룹의 사용자들이 공동으로 관심 있는 웹 콘텐츠의 즐겨 찾기를 공동으로 유지 관리하는 용도로 다차원 벡터형 태그를 제안한다. 제안하는 벡터형 태그는 정보 유용성을 나타내는 색인을 벡터방식으로 기술하고 이것을 활용해 정보의 분류 관리 재활용의 효율을 높이는 표현법이다. 벡터방식 태깅은 대상 키워드에 사용자들이 두 개 이상의 요소에 대한 우선순위를 부여하고 벡터 방식으로 표현한다. 이 때 벡터의 기본이 되는 벡터공간은 정보생성시간, 선호순위 등으로 구성한다. 벡터성분으로 산출할 수 있는 벡터크기가 정보의 유용성을 나타내며 순위측정의 기준이 된다. 제안방식에 의한 순위측정은 단순한 링크구조에 의해 측정된 순위와 비교하였을 때, 사용자의 검색의도에 부합하는 순위 정보를 제공하고 있다.

• Journal of Microbiology and Biotechnology
• /
• 제25권2호
• /
• pp.196-205
• /
• 2015

A Sulfolobus-E. coli shuttle vector for an efficient expression of the target gene in S. acidocaldarius strain was constructed. The plasmid-based vector pSM21 and its derivative pSM21N were generated based on the pUC18 and Sulfolobus cryptic plasmid pRN1. They carried the S. solfataricus P2 pyrEF gene for the selection marker, a multiple cloning site (MCS) with C-terminal histidine tag, and a constitutive promoter of the S. acidocaldarius gdhA gene for strong expression of the target gene, as well as the pBR322 origin and ampicillin-resistant gene for E. coli propagation. The advantage of pSM21 over other Sulfolobus shuttle vectors is that it contains a MCS and a histidine tag for the simple and easy cloning of a target gene as well as one-step purification by histidine affinity chromatography. For successful expression of the foreign genes, two genes from archaeal origins (PH0193 and Ta0298) were cloned into pSM21N and the functional expression was examined by enzyme activity assay. The recombinant PH0193 was successfully expressed under the control of the gdhA promoter and purified from the cultures by His-tag affinity chromatography. The yield was approximately 1 mg of protein per liter of cultures. The enzyme activity measurements of PH0913 and Ta0298 revealed that both proteins were expressed as an active form in S. acidocaldarius. These results indicate that the pSM21N shuttle vector can be used for the functional expression of foreign archaeal genes that form insoluble aggregates in the E. coli system.

• 생명과학회지
• /
• 제14권2호
• /
• pp.315-319
• /
• 2004

사람의 serine palmitoyltransferase(SPT, EC 2.3.1.50)에 대한 항체를 제작하기 위하여 E. coli발현 vector인 pRset vector에 SPTLC1 및 SPTLC2 유전자를 subcloning하고 BL21 (DE3)pLys cell에 발현시켰다. 포유동물의 SPT는 원핵세포의 SPT homodimer와는 달리 SPTLC1 및 SPTLC2 2개의 sub-unit로 된 heterodimer이다. Human embryo kidney cell인 HEK293 cell의 total RNA로부터 RT-PCR을 행하여 cDNA library를 얻은 다음 SPTLC1 및 SPTLC2의 특이적인 primer 들을 이용하여 PCR을 행하였다. SPTLC1 및 SPTLC2 DNA를 hexahistidine fusion 단백질을 발현시킬 수 있는 pRset vector에 cloning하여 pRsetB/SPTLC1 및 pRsetA/SPTLC2를 얻고 염기서열을 확인하였다. 재조합 plasmid를 발현세포인 BL21 cell에 형질전환시킨 다음 ampicillin 및 chroramphenicol 배지에서 선별하여 재조합세포를 얻었다. 1 mM IPTG로서 발현을 유도하였으며 세포 단백질을 SDS-PAGE로 분리한 다음 His-tag antibody로 western blotting을 행하여 SPTLC 및 SPTLC2가 발현되었음을 확인하였다.

• Journal of Microbiology and Biotechnology
• /
• 제24권11호
• /
• pp.1503-1509
• /
• 2014

With the purpose of facilitating the process of stable strain generation, a shuttle vector for integration of genes via a double recombination event into two ectopic sites on the Sulfolobus acidocaldarius chromosome was constructed. The novel chromosomal integration and expression vector pINEX contains a pyrE gene from S. solfataricus P2 ($pyrE_{sso}$) as an auxotrophic selection marker, a multiple cloning site with histidine tag, the internal sequences of malE and malG for homologous recombination, and the entire region of pGEM-T vector, except for the multiple cloning region, for propagation in E. coli. For stable expression of the target gene, an ${\alpha}$-glucosidase-producing strain of S. acidocaldarius was generated employing this vector. The malA gene (saci_1160) encoding an ${\alpha}$-glucosidase from S. acidocaldarius fused with the glutamate dehydrogenase ($gdhA_{saci}$) promoter and leader sequence was ligated to pINEX to generate pINEX_malA. Using the "pop-in" and "pop-out" method, the malA gene was inserted into the genome of MR31 and correct insertion was verified by colony PCR and sequencing. This strain was grown in YT medium without uracil and purified by His-tag affinity chromatography. The ${\alpha}$-glucosidase activity was confirmed by the hydrolysis of $pNP{\alpha}G$. The pINEX vector should be applicable in delineating gene functions in this organism.

• 미생물학회지
• /
• 제33권1호
• /
• pp.1-6
• /
• 1997

5-Enolpyruvylshikimate 3-phosphate(EPSP) synthase는 방향족 아미노산을 생합성하는 shikimate phathway의 6번째 효소로 광범위 제초제인 glyphosate의 target enzyme이다. 본 연구에서는, glyphosate에 저해를 받지 않는 EPSP synthase를 개발하고자 하는 연구의 한 단계로서, 우선, EPSP synthase를 대량 발현시킬수 있는 expression vector인 pET-25b를 사용하여 발현시킨 다음, 발현된 효소가 periplasmic space로 transport되는지 또 발현된 단백질이 효소 활성을 가지고 있는지 확인하고자 하였다. 그 결과, pelB leader를 앞에 붙여 발현시킨 EPSP synthase는 periplasmic space로 제대로 transport되며, 단백질 생산 및 periplasmic space로의 수송은 induction 온도에 의해 크게 좌우된다는 것을 관찰하였다. Periplasmic space로 수송되는 EPSP synthase의 양은 $34^{\circ}C$에서 induction시켰을 때 가장 많은 것으로 나타났다. 한편, pET-25b를 이용하여 발현시킨 EPSP synthase는 C-terminal 부위에 HSV-tag, His-tag등 26개 아미노산이 더 있는 상태로 만들어지는데, His-tag은 $Ni^{2+}$-affinity chromatography를 통한 정제에, HSV-tag은 Western blotting을 통한 detection에 각각 이용할 수 있다. 또한, 이와 같이 발현된 recombinant EPSP synthase는 phosphocellulose resin에 결합하였다가 기질인 shikimate 3-phosphate와 phosphoenolpyruvate에 의해 elution되며, glyphosate에 의해 저해되는등 wildtype효소와 같은 효소 특성을 보였다.

• 대한의생명과학회지
• /
• 제8권3호
• /
• pp.107-113
• /
• 2002

Enterohemorrhagic Escherichia coli (EHEC) strains of serotype O157:H7 have been shown to colonize the intestinal epithelial cell by the attaching and effacing (AE) mechanism. The AE lesion is mediated by an intimin, of which production and expression are controlled by a 3-Kb eaeA gene located EHEC chromosomal DNA. If the eaeA gene is mutated, EHEC O157:H7 strains lose capacity of adhesion to intestinal epithelial cells. In this study, a 891 bp of the 3'-end region of a gamma intimin was amplified by polymerase chain reaction (PCR). The PCR product was inserted into pSTBlue-1 cloning vector and transformed into DE3 (BL21) competent cell. After plasmid mini-preparation and restriction enzyme digestion of eaeA/891-pSTBlue-1 vector, target eaeA gene was re-inserted into pET-28a expression vector and was transformed. Then the expression of recombinant eaeA/891 (891 bp) gene was induced by isopropyl-$\beta$-D-thiogalactopyranoside (IPTG). The expression of the 40-KDa recombinant protein was identified in SDS-PAGE and confirmed by immunoblotting using the His.Tag$^{\circledR}$ and T$_{7}$.Tag$^{\circledR}$ monoclonal antibody. This recombinant protein expressed by eaeA gene could be applied in further studies on the mechanisms of E. coli O157:H7 infection and the development of recombinant vaccine.

• 정보처리학회논문지D
• /
• 제12D권3호
• /
• pp.439-446
• /
• 2005

보다 효과적인 색인어 추출 및 색인어 가중치 결정을 위하여 문서의 내용뿐만 아니라 구조를 이용하여 색인을 추출하는 연구가 이루어지고 있다. 이러한 연구들 대부분이 XML 태그의 중요도가 아닌, 문맥상의 단락에 대한 중요도를 계산하거나 HTML 문서 태그의 중요도를 결정하는 연구들이다. 이러한 기존 연구들은 대부분이 객관적인 실험을 통해서 중요도를 입증하기보다는 상식적인 관점에서 단순한 수치로 중요도를 결정하고 있다. 본 논문에서는 웹 문서 관리를 위한 표준으로 자리잡아가고 있는 XML 문서의 태그 정보를 이용한 자동색인을 위하여, 논문을 구성하는 주요 태그의 가중치를 계산하는 방법을 제안한다. 보다 객관적인 가중치 결정을 위하여 사용자의 질의에 바탕을 둔 사용자의 검색 행위를 반영한다. 그리고 기존 방법을 적용하여 계산된 색인어 가중치를 이용한 검색성능과 비교함으로써 본 논문에서 제안한 방법을 적용하여 계산된 색인어 가중치의 효과를 검증한다.

• 한국멀티미디어학회논문지
• /
• 제23권3호
• /
• pp.440-446
• /
• 2020

The application field of smart terminals is increasing and its application is also spreading in the defense field. The use of smart terminal based map application is very important in battle fields. The problem is that the communication infrastructure is easy to collapse and the use of GPS is usually disturbed. In this paper, we studied the maps stored in the QR tag at the battle field. The problem is to abstract the map information so that it can be stored in the small QR tag. We have abstracted path information on a vector basis and require only a small amount of data compared to imaged path information. We analyzed the amount of data generated by the abstraction and mathematically analyzed the boundary where the amount does not exceed the capacity limit of the QR tag. Our research can be applied not only to battlefields, but also to disaster / disaster scenes, or in environments with difficult Internet communications, such as mountainous areas.

• Journal of Microbiology and Biotechnology
• /
• 제16권10호
• /
• pp.1583-1590
• /
• 2006

T4 endonuclease V (T4 endo V) [EC 3. 1. 25. 1], found in bacteriophage T4, is responsible for excision repair of damaged DNA. The enzyme possesses two activities: a cyclobutane pyrimidine dimer DNA glycosylase (CPD glycosylase) and an apyrimidic/apurinic endonuclease (AP lyase). T4 denV (414 bp cDNA) encoding T4 en do V (138 amino acid) was synthesized and expressed using either an expression vector, pTriEx-4, in E. coli or a baculovirus AcNPV vector, pBacPAK8, in insect cells. The recombinant His-Tag/T4 endo V (rHis-Tag/T4 endo V) protein expressed from bacteria was purified using one-step affinity chromatography with a HiTrap Chelating HP column and used to make rabbit anti-His-Tag/T4 endo V polyclonal antibody for detection of recombinant T4 endo V (rT4 endo V) expressed in insect cells. In the meantime, the recombinant baculovirus was obtained by cotransfection of BacPAK6 viral DNA and pBP/T4 endo V in Spodoptera frugiperda (Sf21) insect cells, and used to infect Sf21 cells to overexpress T4 endo V protein. The level of rT4 endo V protein expressed in Sf21 cells was optimized by varying the virus titers and time course of infection. The optimal expression condition was set as follows; infection of the cells at a MOI of 10 and harvest at 96 h post-infection. Under these conditions, we estimated the amount of rT4 endo V produced in the baculovirus expression vector system to be 125 mg/l. The rT4 endo V was purified to homogeneity by a rapid procedure, consisting of ion-exchange, affinity, and reversed phase chromatographies, based on FPLC. The rT4 endo V positively reacted to an antiserum made against rHis-Tag/T4 endo V and showed a residual nicking activity against CPD-containing DNA caused by UV. This is the first report to have T4 endo V expressed in an insect system to exclude the toxic effect of a bacterial expression system, retaining enzymatic activity.