• BMB Reports
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• 제33권4호
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• pp.294-299
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• 2000

Proteolytic processing of the dengue virus serotype 2 polyprotein precursor is catalyzed by a host signal peptidase and a virus encoded two-component protease consisting of the nonstructural proteins, NS2B and NS3. We expressed in Escherichia coli the NS2B, NS3(pro) and NS2B-NS3 proteins from the dengue virus type 2 strain 16681 as N-terminal fusions with a hexahistidine affinity tag under the control of the inducible trc promoter. All fusion proteins were purified to >90% purity by detergent extraction of inclusion bodies and a single step metal chelate chromatography. Proteins were refolded on-column and recovered with yields of 0.5, 6.0 and 1.0 mg/l of E. coli culture that was grown to $OD_{600}=1.0$ for NS2B, NS3(pro) and NS2B-NS3, respectively. Purified proteins gave strong signals in Western blots using $Ni^{2+}-nitrilotriacetic$ acid as a probe for the presence of the polyHis tag. During the purification process, $(His)_{6}NS2B-NS3$ was apparently not autoproteolytically cleaved at the NS2B/NS3 site.

• 미생물학회지
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• 제52권4호
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• pp.495-499
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• 2016

본 연구에서는 발광 세균에 존재하는 LuxG 단백질의 효소학적 성질을 알아내기 위하여 Photobacterium leiognathi ATCC 25521의 luxG 유전자를 중합효소연쇄반응으로 증폭시켜 T5 프로모터와 6X His-tag 시스템을 지닌 pQE 벡터에 삽입시킨 재조합플라스미드를 제조하여 대장균에 형질전환 후 과발현시켜 단백질을 분리, 정제 하였다. 정제된 단백질의 효소학적 실험 결과, 이 단백질은 FMN과 NADPH 기질에 대한 ferric iron reductase의 기능을 갖고 있음을 확인하였으며 이들 기질에 대한 효소 활성도 상수 $K_m$ 및 $V_{max}$ 값을 결정하였다.

• 대한두경부종양학회지
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• 제36권2호
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• pp.17-20
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• 2020

Background: Compared to the frequency of occurrence of pre-auricular skin tag, cervical chondrocutaneous branchial remnants is one of congenital, benign neck masses that is very rare all over the world. Most of these rare anomalies are reported in case reports and especially, rare cases of unilateral cervical chondrocutaneous branchial remnants have been reported in Korean. Materials & Methods: A 9-year-old male patient visited the hospital on September 2017 for a rod-shaped mass. As a simultaneous diagnosis and treatment method, complete surgical excision was executed. Results: Excised mass was 0.5cm in diameter, 1.2cm in. Histologically, a hyaline and elastic cartilage was found in the core. As a family history, the same remnant was found in the right Anterior neck area of his mother. Conclusion: In this case, it is possible to diagnose and treat simply at the same time and even an optimal aesthetic result can be obtained.

• 대한의생명과학회지
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• 제8권3호
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• pp.107-113
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• 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.

• 한국미생물·생명공학회지
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• 제46권3호
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• pp.244-252
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• 2018

Protein disulfide isomerase A3 (PDIA3) is a major member of the protein disulfide isomerase (PDI) family. PDI proteins commonly reside in the endoplasmic reticulum and mediate important thiol-disulfide interchanges during post-translational protein folding. Unlike other PDI family members, PDIA3 is ubiquitous in various organ systems. However, its physiological activity varies in other tissues. PDIA3 has been associated with cancer, airway inflammation, neurodegenerative diseases, and metabolic diseases. However, the mechanisms of the association of PDIA3 with these pathological conditions remain unclear. Recombinant PDIA3 (rPDIA3) is needed to clarify the interactions between PDIA3 and certain physiological phenomena. In the present study, we aimed to produce highly purified rPDIA3 for use in pathological experiments. We expressed rPDIA3 with a histidine-enriched elongated peptide tag in Escherichia coli and obtained rPDIA3 at 97.8% purity using consecutive His-tag and reverse-phase chromatography. Elongated peptide tags screened from artificially designated library had dual functions for protein expression and simple purification.

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

• KSBB Journal
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• 제18권4호
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• pp.306-311
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• 2003

융합단백질의 절단을 위해 EK를 고정화하여 액상 절단반응과 같은 80％의 절단수율을 얻을 수 있었다. 그리고 니켈 친화칼럼을 이용하여 간단한 정제공정을 구축하였다. 공유결합한 EK의 경우 니켈친화 결합한 EK보다 높은 재접힘 수율을 나타내었고 풀림과 재접힘을 이용하여 효소의 초기 활성을 회복함에 따라서 반복사용을 통한 경제적인 절단공정을 구축할 수 있게 되었다. 그러나 고정화 과정에서 효소의 활성이 감소하는 문제점과 고정화 수율을 높이기 위한 연구가 필요하다.

• Journal of Microbiology and Biotechnology
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• 제31권11호
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• pp.1583-1590
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• 2021

Studies have demonstrated that PE_PGRS45 is constitutively expressed under various environmental conditions (such as nutrient depletion, hypoxia, and low pH) of the in vitro growth conditions examined, indicating that PE_PGRS45 protein is critical to the basic functions of Mycobacterium tuberculosis. However, there are few reports about the biochemical function and pathogenic mechanism of PE_PGRS45 protein. The fact that this M. tuberculosis gene is not easily expressed in E. coli may be mainly due to the high content of G+C and the use of unique codons. Fusion tags are indispensable tools used to improve the soluble expression of recombinant proteins and accelerate the characterization of protein structure and function. In the present study, His6, Trx, and His6-MBP were used as fusion tags, but only MBP-PE_PGRS45 was expressed solubly. The purification using His6-MBP tag-specific binding to the Ni column was easy to separate after the tag cleavage. We used the purified PE_PGRS45 to immunize New Zealand rabbits and obtained anti-PE_PGRS45 serum. We found that the titer of polyclonal antibodies against PE_PGR45 was higher than 1:256000. The result shows that purified PE_PGRS45 can induce New Zealand rabbits to produce high-titer antibodies. In conclusion, the recombinant protein PE_PGRS45 was successfully expressed in E. coli and specific antiserum was prepared, which will be followed by further evaluation of these specific antigens to develop highly sensitive and specific diagnostic tests for tuberculosis.

• 한국지능시스템학회논문지
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• 제17권6호
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• pp.773-779
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• 2007

인터넷 게시판은 수많은 사용자들의 생각과 그들이 가진 정보를 교환하기 위하여 주요한 방법으로 사용되고 있다. 그러나 공동의 게시판은 사용자 개개인의 관심분야를 만족시키지 못한다. 본 연구에서는 웹 2.0을 활용하여 각각의 사용자에게 맞춤형 서비스를 제공하는 인터넷 게시판을 설계한다. 설계될 인터넷 게시판은 사용자에게 제공되는 정보는 동일하지만, 각 사용자마다 설정된 정보에 의하여 정보의 분류가 다르게 이루어지도록 하여 자신이 원하는 정보를 보다 빠르게 검색할 수 있도록 하였다. 또한, 각 사용자는 개인 게시판을 생성하여 모든 사용자가 공유하는 게시판에서 자신에게 필요한 정보만을 자동으로 수집하여 저장할 수 있으며, 외부 RSS 피드들을 필터링하여 개인 게시판에 연결하거나, 자신만의 정보를 개인 게시판에 등록할 수도 있다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XII)
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• pp.550-553
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• 2003

$Hisx_6-GFPuv-Cyt$ c fusion protein을 E. coli 균주 JM109과 BL21 각각에서 발현시킨 결과 발현온도는 $30^{\circ}C$보다 $37^{\circ}C$에서, BL21보다 JM109에서의 발현량이 더 많았다. 그러나 JM109, $37^{\circ}C$에서 발현시킨 fusion protein의 $Ni^{2+}-IDA-agarose$ purification결과 약 45kDa 부근의 fusion protein의 density가 감소되었음을 SDS-PAGE analysis을 통해 알 수 있었다. 또한 western blotting analysis를 통해 이 impurity가 degraded fusion임을 확인 할 수 있었다. degraded fusion은 BL21 균주에서 발현시킨 fusion protein에서도 생성됨을 확인하였다. 모든 결과를 종합해 볼때 $Hisx_6-GFPuv-Cyt$ c fusion protein의 발현은 IM109, $37^{\circ}C$에서 더 많았지만, BL21, $37^{\circ}C$에서 expression시킨 fusion protein이 보다 안정하다고 판단 되어진다. 향후 fusion protein이 bioelectronic device에 적용되려면 degraded fusion protein의 생성을 줄여 activity를 유지하도록 안정한 형태로 발현되어 순수하게 분리정제 되어야 하겠다.