• Molecules and Cells
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• 제21권3호
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• pp.376-380
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• 2006

Gene expression is regulated in large part at the level of transcription under the control of sequence-specific transcriptional regulatory proteins. Therefore, the ability to affect gene expression at will using sequencespecific artificial transcription factors would provide researchers with a powerful tool for biotechnology research and drug discovery. Previously, we isolated 56 novel sequence-specific DNA-binding domains from the human genome by in vivo selection. We hypothesized that these domains might be more useful for regulating gene expression in higher eukaryotic cells than those selected in vitro using phage display. However, an unpredictable factor, termed the "context effect", is associated with the construction of novel zinc finger transcription factors--- DNA-binding proteins that bind specifically to 9-base pair target sequences. In this study, we directly selected active artificial zinc finger proteins from a zinc finger protein library. Direct in vivo selection of constituents of a zinc finger protein library may be an efficient method for isolating multi-finger DNA binding proteins while avoiding the context effect.

• Journal of Information Processing Systems
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• 제8권3호
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• pp.459-470
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• 2012

Recently, high-throughput technologies such as the two-hybrid system, protein chip, Mass Spectrometry, and the phage display have furnished a lot of data on protein-protein interactions (PPIs), but the data has not been accurate so far and the quantity has also been limited. In this respect, computational techniques for the prediction and validation of PPIs have been developed. However, existing computational methods do not take into account the fact that a PPI is actually originated from the interactions of domains that each protein contains. So, in this work, the information on domain modules of individual proteins has been employed in order to find out the protein interaction relationship. The system developed here, WASPI (Web-based Assistant System for Protein-protein interaction Inference), has been implemented to provide many functional insights into the protein interactions and their domains. To achieve those objectives, several preprocessing steps have been taken. First, the domain module information of interacting proteins was extracted by taking advantage of the InterPro database, which includes protein families, domains, and functional sites. The InterProScan program was used in this preprocess. Second, the homology comparison with the GO (Gene Ontology) and COG (Clusters of Orthologous Groups) with an E-value of $10^{-5}$, $10^{-3}$ respectively, was employed to obtain the information on the function and annotation of each interacting protein of a secondary PPI database in the WASPI. The BLAST program was utilized for the homology comparison.

• Journal of Microbiology and Biotechnology
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• 제28권12호
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• pp.2113-2120
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• 2018

Cross-reactive material 197 ($CRM_{197}$) is a non-toxic mutant of diphtheria toxin containing a single amino acid substitution of glycine 52 with glutamic acid. $CRM_{197}$ has been used as a carrier protein for poorly immunogenic polysaccharide antigens to improve immune responses. In this study, to develop a sandwich ELISA that can detect $CRM_{197}$ and $CRM_{197}$ conjugate vaccines, we generated a human anti-$CRM_{197}$ monoclonal antibody (mAb) 3F9 using a phage-displayed human synthetic Fab library and produced mouse anti-$CRM_{197}$ polyclonal antibody. The affinity ($K_D$) of 3F9 for $CRM_{197}$ was 3.55 nM, based on Bio-Layer interferometry, and it bound specifically to the B fragment of $CRM_{197}$. The sandwich ELISA was carried out using 3F9 as a capture antibody and the mouse polyclonal antibody as a detection antibody. The detection limit of the sandwich ELISA was <1 ng/ml $CRM_{197}$. In addition, the 3F9 antibody bound to the $CRM_{197}$-polysaccharide conjugates tested in a dose-dependent manner. This ELISA system will be useful for the quantification and characterization of $CRM_{197}$ and $CRM_{197}$ conjugate vaccines. To our knowledge, this study is the first to generate a human monoclonal antibody against $CRM_{197}$ and to develop a sandwich ELISA for $CRM_{197}$ conjugate vaccines.

• BMB Reports
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• 제36권5호
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• pp.493-498
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• 2003

The scFv antibody towards the Burkholderia pseudomallei exotoxin was previously constructed by phage display and exhibited good specificity towards the exotoxin. We report here the optimization of the scFv expression in an E. coli expression system. Four different E. coli strains (ER2537, TG1, HB2151, and XL1-Blue) were examined for optimal expression of the scFv protein. Two types of carbon source (i.e. 0.2% glucose and 0.2% glycerol) were also tested for their ability to induce the scFv expression. Cells that carried the scFv construct were grown at $30^{\circ}C$ and induced with 0.05 mM IPTG. The expression was then monitored by SDS-PAGE, Western blotting, and indirect ELISA. The Western blot profile showed different levels of the scFv expression among the host strains; XL1-Blue exhibited the highest level of the scFv protein expression. Glycerol at a concentration of 0.2% (v/v) significantly increased the scFv protein expression level when compared to 0.2% (w/v) glucose. Further optimization demonstrated that the scFv protein expression in XL1-Blue was the most optimal with a glycerol concentration as low as 0.05%. However, by indirect ELISA, only the scFv protein that was expressed in 0.2% (v/v) glycerol exhibited high specificity towards the Burkholderia pseudomallei exotoxin.

• 한국동물위생학회지
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• 제46권4호
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• pp.325-334
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• 2023

Edema disease (ED) in pigs is enterotoxemia caused by Shiga toxin type 2e (Stx2e)-producing Escherichia coli (STEC) and frequently occurs in young piglets. Therefore, ED causes enormous economic losses in pig farms. In this study, a modified Stx2e (mStx2e) antigen was expressed and purified using commercial E. coli expression system. Monoclonal antibody was serviced by Ynto Ab Inc., using Phage Display Technique. Anti-Stx2e antibodies in piglets were measured by indirect ELISA using mStx2e antigens. Naive Stx2e in piglets were detected by Sandwich ELISA using Stx2e-monoclonal antibodies and commercial Stx2e-polyclonal antibodies. Among 3,480 piglets, anti-Stx2e antibodies were observed in 2,573 piglets. The 49.4% among 830 piglet serum samples possessed 0.625 ㎍/mL or more of Stx2e proteins. The 18.3% of 830 sera had 0.313 ㎍/mL of Stx2e proteins. The 32.3% of 830 samples held 0.156 ㎍/mL or less of Stx2e proteins. These results show that indirect ELISA using mStx2e antigen and Sandwich ELISA using Stx2e-monoclonal and polyclonal antibodies can be useful to detect ED in piglets.

• 한국잠사곤충학회지
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• 제53권1호
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• pp.44-49
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• 2015

본 연구는 누에 후부실샘에서 특이적으로 발현하는 새로운 전사체를 탐색하고 이의 프로모터 영역을 분석함으로서 향후 형질전환누에 제작용 전이벡터 시스템의 효율성 제고를 위해 활용하고자 하였다. 우선 새로운 후부실샘 특이 발현 전사체 선발을 위해 ACP-based dd-PCR 방법으로 후부실샘에서 특이적으로 발현하는 34개 PCR 증폭 산물이 선발되었는데, 이 중 지금까지 보고된 바 없는 새로운 전사체인 ACP-16(366 bp)이 선발되었다. Northern blotting hybridization 분석 결과, ACP-16은 후부실샘에서 특이적으로 발현되는 것이 확인되었으며 전사체 발현량에서는 fibroin light chain 보다는 적었으며 전사체 크기에서는 fibroin light chain 보다는 다소 큰 것으로 확인되었다. ACP-16 유전자 프로모터 영역을 클로닝 하기 위해 게놈 유전자은행으로부터 ACP-16 (366 bp)를 탐침으로 전체 17.4 kb 크기의 파이지 클론을 선발할 수 있었으며, 전사체 상류에 유전자 발현조절에 필요한 TATA box와 Cap box 구조를 확인할 수 있었다. 본 연구에서 확보된 ACP-16 유전자의 프로모터 영역은 이후 코어 프로모터 개발 연구를 통하여 효과적인 누에 형질전환 시스템 구축에 적용할 수 있을 것으로 기대된다.