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Yeast two-hybrid 시스템을 통한 K11 phage lysozyme과 K11 phage RNA 중합효소와의 결합에 대한 연구
전현정,이상수,Junn, Hyun-Jung,Lee, Sang-Soo 배재대학교 자연과학연구소 2004 自然科學論文集 Vol.14 No.2
박테리오 파이지 K11 lysozyme은 최근에 우리 실험실에서 클로닝 되었으며, 숙주균주의 세포벽을 분해하는 고유의 lysozyme활성과 박테리오 파아지 K11 RNA 중합효소의 전사반응을 억제하는 활성을 가지고 있는 것으로 확인되었다. 이미 잘 연구된 박테리오 파아지 T7 lysozyme의 경우 클로닝되고 분리 정제된 T7 lysozyme 단백질의 3차 구조 및 T7 RNA 중합효소와의 결합양상에 대하여 밝혀졌다. 따라서 우리 실험실에서는 K11 lysozyme과 K11 RNA 중합효소와의 결합 정도 및 그 특성을 파악할 목적으로 yeast two hybrid 시스템을 통하여 K11 RNA 중합효소와 K11 lysozyme의 단백질-단백질 상호작용을 알아보고자 하였다. LexA 시스템을 이용하여 LexA DNA 결합 부위를 갖고 있는 pLexA에 K11 lysozyme 유전자를 삽입하여 prey로 하였따. 활성 부위로는 B42 융합 단백질을 만드는 pJG4-5에 K11 RNA 중합효소의 결합은 생체 밖에서 reporter 유전자인 lacZ와 leu2의 발현으로 확인되었으며 이들의 결합정도와 결합부위에 대한 연구들은 진행중에 있다. Recently phage K11 lysozyme was cloned and characterized in our lab. The K11 lysozyme was identified to have dual functions. It not only cuts a peptidoglycan bond in bacterial cell wall but also acts as an inhibitor of K11 RNA polymerase. It has been known that the T7 lysozyme binds specifically to T7 RNA polymerase and inhibits transcription. The dual activities of K11 lysozyme are atreeable to the case of T7 phage lysozyme and RNA polymerare. In order to identify the binding magnitude of K11 lysozyme with K11 RNA polymerase, yeast two-hybrid system was used. K11 phage lysozyme gene was introduced into pLexA plasmid and used as a prey. Also, K11 phage RNA polymerase gene was introduced into pJG4-5 and used as a bait. The binding between K11 lysozyme and K11 RNA polymerase was demonstrated by expression of reporter genes such as lacZ and leu2.
Dongwoo Lee,Jida Liu,Hyun Jung Junn,Eun-Joo Lee,Kyu-Shik Jeon,Dai-Wu Seol 생화학분자생물학회 2019 Experimental and molecular medicine Vol.51 No.-
Gene therapy is emerging as an effective treatment option for various inherited genetic diseases. Gutless adenovirus(GLAd), also known as helper-dependent adenovirus (HDAd), has many notable characteristics as a gene deliveryvector for this particular type of gene therapy, including broad tropism, high infectivity, a large transgene cargocapacity, and an absence of integration into the host genome. Additionally, GLAd ensures long-term transgeneexpression in host organisms owing to its minimal immunogenicity, since it was constructed following the deletion ofall the genes from an adenovirus. However, the clinical use of GLAd for the treatment of inherited genetic diseases hasbeen hampered by unavoidable contamination of the highly immunogenic adenovirus used as a helper for GLAdproduction. Here, we report the production of GLAd in the absence of a helper adenovirus, which was achieved with ahelper plasmid instead. Utilizing this helper plasmid, we successfully produced large quantities of recombinant GLAd. Importantly, our helper plasmid-based system exclusively produced recombinant GLAd with no generation of helperplasmid-originating adenovirus and replication-competent adenovirus (RCA). The recombinant GLAd that wasproduced efficiently delivered transgenes regardless of their size and exhibited therapeutic potential for Huntington’sdisease (HD) and Duchenne muscular dystrophy (DMD). Our data indicate that our helper plasmid-based GLAdproduction system could become a new platform for GLAd-based gene therapy.