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김외연,이훈실,서숙재,조무제,이상열,김재원,Kim, Woe-Yeon,Lee, Hoon-Sil,Suh, Sook-Jae,Cho, Moo-Je,Lee, Sang-Yeol,Kim, Jae-Won The Microbiological Society of Korea 1994 미생물학회지 Vol.32 No.2
Serratia marcescens가 세포외로 분비하는 nuclease의 유전자가 발현된 Escherichia coli JM107을 배양하여 다량의 효소를 정제하였다. Matrex green gel과 heparin agarose gel column chromatography법으로 약 50배 정제한 효소는 분자량이 29KDa였으며, 전기영동 상에서 단일 띠를 보였다. 이 단백질을 이용하여 polyclonal antibody를 만들고, 면역조직화학법으로 세포내의 분포를 조사하였다. Nuclease는 주로 세포막에 존재하였고, 이를 토대로 효소가 세포질에서 합성된 후 세포막으로 빠르게 이동함을 알 수 있었다. 이 결과는 세포의 막분획에서 효소의 활성의 대부분이 회수되며, 면역블럿 방법으로 효소의 대부분이 세포막에서 검출된다는 결과와 일치하였다. Nuclease was secreted to the environmental media from the Escherichia coli JM107 tranformant harboring the extracellular nuclease gene of Serratia marcescens in the plasmid of pNUC4. Under the growth conditions, the amount of secreted enzyme was increased in parallel with bacterial growth conditions, the amount of secreted enzyme was increased in parallel with bacterial growth. The enzyme was purified using chromatofraphic procedures of Matrex green gel and heparin agarose affinity gel, resulted in 50-fold purification with 15% recovery of the enzyme. The apparent molecular weight of the enzyme was estimated to be 29Kda by sodium dodecylsulfate denaturing gel electrophoresis. Using the purified enzyme, polyclonal antibody was obtained from the rabbit. The specificity of the antibody was confirmed by immunoblotting and immunoprecipitaion. For the investigation of cellular distribution of the enzyme, cells were fractionated into three fractions; cytoplasm, periplasm and extracellular fluid. While more than 80% of the enzymatic activity was detected in the extracellular fluid and periplasm, a little was found in the cytoplasm, indicating that the enzyme was likely to be immediately exported to the membrane for excretion after biosynthesis. These results were confirmed again by immunocytochemistry technique using the antibody.
SUMO and SUMOylation in Plants
박희진,김외연,박형철,이상열,한스,윤대진 한국분자세포생물학회 2011 Molecules and cells Vol.32 No.4
The traditional focus on the central dogma of molecular biology, from gene through RNA to protein, has now been replaced by the recognition of an additional mechanism. The new regulatory mechanism, post-translational modifications to proteins, can actively alter protein function or activity introducing additional levels of functional complexity by altering cellular and sub-cellular location, protein interactions and the outcome of biochemical reaction chains. Modifications by ubiquitin (Ub) and ubiquitin-like modifiers systems are conserved in all eukaryotic organisms. One of them, small ubiquitin-like modifier (SUMO) is present in plants. The SUMO mechanism includes several isoforms of proteins that are involved in reactions of sumoylation and de-sumoylation. Sumoylation affects several important processes in plants. Outstanding among those are responses to environmental stresses. These may be abiotic stresses, such as phosphate deficiency, heat, low temperature, and drought, or biotic stressses, as well including de-fense reactions to pathogen infection. Also, the regula-tions of flowering time, cell growth and develop-ment, and nitrogen assimilation have recently been added to this list. Identification of SUMO targets is material to characterize the function of sumoylation or desumoylation. Affinity purification and mass spectrometric identification have been done lately in plants. Further SUMO non-covalent binding appears to have function in other model organisms and SUMO interacting proteins in plants will be of interest to plant biologists who dissect the dynamic function of SUMO. This review will discuss results of recent insights into the role of sumoylation in plants.
Laila Khaleda,차준영,김민갑,김외연 한국식물학회 2017 Journal of Plant Biology Vol.60 No.6
Arabidopsis GIGANTEA (GI) is encoded by asingle gene and highly conserved among vascular plants andits mutants display pleiotropic phenotypes involved indiverse biological processes such as light signaling, circadianclock, and sucrose metabolism as well as abiotic stressresponses. However, molecular mechanisms of GI arelargely unknown due to the lack of useful antibody. To date,the epitope tags have been widely used to detect GI in plants,but it needs to generate the transgenic plants which take afew months. Here, we produced polyclonal α-GI antibodyusing truncated variants of GI having amino-terminal (1-858aa) and carboxyl-terminal (920-1173) regions as antigens. Both recombinant His-GI1-858 and His-GI920-1173 proteins wereindividually and successfully expressed in E. coli andimmunized into rabbit. Anti-serum was purified by antigenspecificaffinity purification method using both recombinantHis-GI1-858 and His-GI920-1173 proteins. Purified polyclonal α-GI antibody not only detected endogenous GI proteins inwild-type Arabidopsis plants, but also reenacted its dieloscillations. Furthermore, the antibody showed cross-reactivitywith the GI orthologs in other plants such as Chinesecabbage, rape and tomato. Our polyclonal GI antibody couldhelp to determine the molecular mechanisms of GI involvedin largely unknown pleiotropic responses in plants.