• Journal of Plant Biotechnology
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• 제45권4호
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• pp.315-321
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• 2018

Molecular and functional characterization of proteins and their levels is of great interest in understanding the mechanism of diverse cellular processes. In this study, we report on the convenient Escherichia coli-based protein expression system that allows recombinant of soluble proteins expression and cytosolic domain of membrane-localised kinases, followed by the detection of autophosphorylation activity in protein kinases. This approach is applied to regulatory proteins of Arabidopsis thaliana, including 14-3-3, calmodulin, calcium-dependent protein kinase, TERMINAL FLOWER 1(TFL1), FLOWERING LOCUS T (FT), receptor-like cytoplasmic kinase and cytoplasmic domain of leucine-rich repeat-receptor like kinase proteins. Our Western blot analysis which uses phospho-specific antibodies showed that five putative LRR-RLKs and two putative RLCKs have autophosphorylation activity in vitro on threonine and/or tyrosine residue(s), suggesting their potential role in signal transduction pathways. Our findings were also discussed in the broader context of recombinant expression and biochemical analysis of soluble and membrane-localised receptor kinases in microbial systems.

• 대한화장품학회:학술대회논문집
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• 대한화장품학회 2003년도 IFSCC Conference Proceeding Book I
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• pp.630-644
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• 2003

A human Cu, Zn-superoxide dismutase (Cu, Zn-SOD) was fused with a Tat PTD of HIV-1 to produce a novel anti-aging ingredient, Tat-SOD for cosmeceuticals. Test of stability and evaluation of transduction efficacy and enzymatic activity suggest Tat-SOD is an effective active ingredient for anti-aging treatment.

• Macromolecular Research
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• 제16권6호
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• pp.481-488
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• 2008

Apoptosis is a natural cell suicide mechanism to maintain homeostasis. However, many of the diseases encountered today are caused by aberrant apoptosis where excessive apoptosis leads to neurodegenerative disorders, ischemic heart disease, autoimmune disorders, infectious diseases, etc. A variety of antiapoptotic agents have been reported to interfere with the apoptosis pathway. These agents can be potential drug candidates for the treatment or prevention of diseases caused by dysregulated apoptosis. Obviously, world-wide pharmaceutical and biotechnology companies are gearing up to develop antiapoptotic drugs with some products being commercially available. Polymeric drug delivery systems are essential to their success. Recent R&D efforts have focused on the chemical or bioconjugation of antiapoptotic proteins with the protein transduction domain (PTD) for higher cellular uptake with antibodies for specific targeting as well as with polymers to enhance the protein stability and prolonged effect with success observed both in vivo and in vitro. All these different fusion antiapoptotic proteins provide promising results for the treatment of dysregulated apoptosis diseases.

• Molecules and Cells
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• 제21권1호
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• pp.104-111
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• 2006

Gene therapy with nonviral vectors using the suicide gene/prodrug activating system of herpes simplex virus type-1 thymidine kinase (HSV1-TK)/ganciclovir (GCV) is inefficient in killing malignant tumor cells due to two major factors: (a) an unsatisfactory bystander effect; (b) short-lived expression of the protein. To study the capacity of the protein transduction domain (PTD) of HIV-1 TAT protein to enhance HSV1-TK/GCV cancer gene therapy, we constructed three fusion proteins TAT-TK, TK-TAT and TK. TAT-TK retained as much enzyme activity as TK, whereas that of TK-TAT was much lower. TAT-TK can enter HepG2 cells and much of it is translocated to the nucleus. The transduced HepG2 cells are killed by exogenously added GCV and have bystander effects on untransduced HepG2 cells. Most importantly, the introduced recombinant protein is stable and remains functional for several days at least, probably because nuclear localization protects it from the cytoplasmic degradation machinery and provides access to the nuclear transcription machinery. Our results indicate that TAT fusion proteins traffic intercellularly and have enhanced stability and prodrug cell killing activity. We conclude that TAT has potential for enhancing enzyme prodrug treatment of liver cancers.

• 한국진공학회:학술대회논문집
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• 한국진공학회 2013년도 제44회 동계 정기학술대회 초록집
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• pp.84-84
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• 2013

FcBP consisting of 13 amino acids specifically binds to Immunoglobulin G Fc domain. Initially, we utilized this peptide for preparation of antibody chip as a PEG composite for enhanced solubility. After then, the peptide conjugate was immobilized on agarose resin, resulting in highly efficient affinity column for antibody purification. The efficiency was comparable to commercial Protein A column. Recently, this peptide was conjugated with cell penetratingpeptide (CPP) on a backbone of GFP, affording antibody transducer, which carries antibody into live cells by simple mixing of antibody and the transducer in cell culture media. Antibody transduction into cells was monitored by live cell imaging. More recently, the FcBP was fused to ferritin cage, which consists of 24 ferritin protein molecules. The FcBP-ferritin cage showed greatly increased binding affinity to human IgG. Its binding was analyzed by QCM and SPR analysis. Finally, it was selectively delivered by Herceptin to SKBR3, a breast cancer cell, over MCF10A, non-tumorigenic cells.

• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
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• pp.81.2-82
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• 2003

Plants have evolved along with pathogens, and they have developed sophisticated defense systems against specific microorganisms to survive. G-protons are considered one of the upstream signaling components working as a key for the defense signal transduction pathway. For activation and inactivation of G-protein, GTP-biding proteins are involved. GTP -binding proteins are found in all organisms. Small GTP-binding proteins, having masses of 21 to 30kD, belong to a superfamily, often named the Ras supefamily because the founding members are encoded by human Ras genes initially discovered as cellular homologs of the viral ras oncogene. Members of this supefamily share several common structural features, including several guanine nucleotide binding domains and an effector binding domain. However, exhibiting a remarkable diversity in both structure and function. They are important molecular switches that cycle between the GDP-bound inactive form into the GTP-bound active form through GDP/GTP replacement. In addition, most GTP-binding proteins cycle between membrane-bound and cytosolic forms. such as the RAC family are cytosolic signal transduction proteins that often are involved in processing of extracellular stimuli. Plant RAC proteins are implicated in regulation of plant cell architecture secondary wall formation, meristem signaling, and defense against pathogens. But their molecular mechanisms and functions are not well known. We isolated a RacB homolog from rice to study its role of defense against pathogens. We introduced the constitutively active and the dominant negative forms of the GTP-hinging protein OsRacB into the wild type rice. The dominant negative foms are using two forms (full-sequence and specific RNA interference with RacB). Employing southern, and protein analysis, we examine to different things between the wild type and the transformed plant. And analyzing biolistic bombardment of onion epidermal cell with GFP-RacB fusion protein revealed association with the nucle.

• Journal of Periodontal and Implant Science
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• 제38권2호
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• pp.153-162
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• 2008

Purpose: Recombining bone morphogenetic protein (BMP) is usually acquiredfrom high level animals. Though this method is effective, its high cost limits its use. The purpose of this study was to evaluate the effect of bone morphogenetic protein-2 with protein transduction domain (BMP-2/PTD;TATBMP-2) on bone regeneration. Rat calvarial defect model and osteoblastic differentiation model using MC3T3 cell were used for the purpose of the study. Materials and Methods: MC3T3 cells were cultured until they reached a confluence stage. The cells were treated with 0, 0.1, 1, 10, 100, 500 ng/ml of BMP-2/PTD for 21 days and at the end of the treatment, osteoblastic differentiation was evaluated usingvon Kossa staining. An 8mm, calvarial, critical-size osteotomy defect was created in each of 48 male Spraque-Dawley rats (weight $250{\sim}300\;g$). Three groups of 16 animals each received either BMP-2/PTD (0.05mg/ml) in a collagen carrier, collagen only, or negative surgical control. And each group was divided into 2 and 8 weeks healing intervals. The groups were evaluated by histologic analysis(8 animals/group/healing intervals) Result: In osteoblastic differentiation evaluation test, a stimulatory effect of BMP-2/PTD was observed in 10ng/ml of BMP-2/PTD with no observation of dose-dependent manner. The BMP-2/PTD group showed enhanced local bone formation in the rat calvarial defect at 2 weeks. New bone was observed at the defect margin and central area of the defect. However, new bone formation was observed only in 50% of animals used for 2weeks. In addition, there was no new bone formation observed at 8 weeks. Conclusion: The results of the present study indicated that BMP-2/PTD(TATBMP-2) have an positive effect on the bone formation in vitro and in vivo. However, further study should be conducted for the reproducibility of the outcomes.

• Journal of Microbiology and Biotechnology
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• 제16권9호
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• pp.1441-1447
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• 2006

The light perception and phototransformation of phytochromes are the first process of the phytochrome-mediated light signal transduction. The chromophore ligation and its photochromism of various site-specific and deletion mutants of pea phytochrome A and bacterial phytochrome-like protein (Cph1) were analyzed in vitro. Serial truncation mutants from the N-terminus and C-terminus indicated that the minimal N-terminal domain for the chromophore ligation spans from the residue 78 to 399 of pea phytochrome A. Site-specific mutants indicated that several residues are critical for the chromophore ligation and/or photochromism. Histidine-324 appears to serve as an anchimeric residue for photochromism through its H-bonding function. Isoleucine-80 and arginine-383 playa critical role for the chromophore ligation and photochromism. Arginine-383 is presumably involved in the stabilization of the Pfr form of pea phytochrome A. Apparently, the amphiphilic ${\alpha}$-helix centered around the residue-391 is in the chromophore pocket and critical for the chromophore ligation.

• 한국정보통신학회:학술대회논문집
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• 한국정보통신학회 2018년도 춘계학술대회
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• pp.588-591
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• 2018

Baculovirus는 원래 알팔파 루퍼 (looper)로부터 분리되었으며 154 개의 오픈 리딩 프레임 (ORF)을 가진 134-kbp 게놈을 포함하고 있다. 주요 캡시드 단백질 VP39는 약간의 단백질과 함께 p6.9 단백질로 DNA를 감싸는 뉴클레오 캡시드($21nm{\times}260nm$)로 형성된다. 그것들은 막대 모양의 캡시드 안에 이중 가닥의 고리 모양의 슈퍼 코일 DNA 분자이다. 야생형 baculovirus는 용균 및 폐색 된 생명주기를 모두 나타내며 바이러스 복제의 3 단계에 걸쳐 독립적으로 발달한다. 재조합 baculovirus는 광범위한 포유류 세포 유형에서 벡터를 전달하고 재조합 단백질을 발현 할 수 있다. 특히, 이들 baculovirus 벡터에 우세한 선별 마커를 포함시킴으로써 많은 세포에서 다양한 재조합 유전자를 발현시킬 수 있다. 본 연구의 배큘로 바이러스 벡터는 cytomegalovirus (CMV) 프로모터, uroplakin II promoter, polyhedron promoter, 수포 구내염 바이러스 G (VSVG), 녹색 형광 단백질 (EGFP), 단백질 전달 도메인 (PTD) 유전자 등으로 재구성되었다. 이러한 재구성 된 벡터를 다양한 세포 및 세포주에 감염시켰다. 우리는 다른 재조합 벡터와 비교하여 이러한 재조합 벡터의 전이 및 발현을 조사하는 수행하였다. 본 연구에서, 우리는 이 재조합 벡터의 형질 감염 및 발현이 어떤 대조군 벡터보다 더 높은 효능을 갖는다는 것을 알았다. 본 연구는 과학 기술부, 한국 정보 기술 진흥 기금 (MSIP)이 후원하는 한국 연구 재단 (NRF)을 통해 중견 연구원 프로그램 (NRF-2016R1A2B4016552)을 통해 지원되었다.

• BMB Reports
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• 제40권4호
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• pp.562-570
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• 2007

Soluble or cell-bound IL-1 receptor accessory protein (IL-1RAcP) does not bind IL-1 but rather forms a complex with IL-1 and IL-1 receptor type I (IL-1RI) resulting in signal transduction. Synthetic peptides to various regions in the Ig-like domains of IL-1RAcP were used to produce antibodies and these antibodies were affinity-purified using the respective antigens. An anti-peptide-4 antibody which targets domain III inhibited 70% of IL-$1\beta$-induced productions of IL-6 and PGE2 from 3T3-L1 cells. Anti-peptide-2 or 3 also inhibited IL-1-induced IL-6 production by 30%. However, antipeptide-1 which is directed against domain I had no effect. The antibody was more effective against IL-$1\beta$ compared to IL-$1\alpha$. IL-1-induced IL-6 production was augmented by coincubation with PGE2. The COX inhibitor ibuprofen blocked IL-1-induced IL-6 and PGE2 production. These results confirm that IL-1RAcP is essential for IL-1 signaling and that increased production of IL-6 by IL-1 needs the co-induction of PGE2. However, the effect of PGE2 is independent of expressions of IL-1RI and IL-1RAcP. Our data suggest that domain III of IL-1RAcP may be involved in the formation or stabilization of the IL-1RI/IL-1 complex by binding to epitopes on domain III of the IL-1RI created following IL-1 binding to the IL-1RI.