http://chineseinput.net/에서 pinyin(병음)방식으로 중국어를 변환할 수 있습니다.
변환된 중국어를 복사하여 사용하시면 됩니다.
개별검색 DB통합검색이 안되는 DB는 DB아이콘을 클릭하여 이용하실 수 있습니다.
통계정보 및 조사
예술 / 패션
<해외전자자료 이용권한 안내>
- 이용 대상 : RISS의 모든 해외전자자료는 교수, 강사, 대학(원)생, 연구원, 대학직원에 한하여(로그인 필수) 이용 가능
- 구독대학 소속 이용자: RISS 해외전자자료 통합검색 및 등록된 대학IP 대역 내에서 24시간 무료 이용
- 미구독대학 소속 이용자: RISS 해외전자자료 통합검색을 통한 오후 4시~익일 오전 9시 무료 이용
※ 단, EBSCO ASC/BSC(오후 5시~익일 오전 9시 무료 이용)
Background: Phospholipase C-β4 (PLC-β4) is known to be one of the most important signal transducing molecules; however, its biophysical and chemical characteristics are not well known due to the difficulty in purifying PLC-β4 from bovine retina. In the present study, we used the baculovirus expression system in order to express and purify large amounts of PLC-β4. With this system, we also tried to produce chimeric PLC-β3/β4 and PLC-β4/β3 protein in order to study the structure-activity relationship between N terminal and C terminal portion of PLC-βs. Methods: I cloned PLC-β4 to the baculovirus expression system by the polymerase chain reaction method and infected the PLC-β4to Sf9 cells. I purified recombinant PLC-β4 proteins using sequential high performnance liquid chromatography (HPLC) by using the TSK phenyl-5PW column and the TSK heparin-5PW column. With this similar method, I was able to express chimeric PLC-β3/β4 and PLC-β4/β3 proteins. Results: With the two step HPLC, I was able to purify PLC-β4 by 30-fold; this purified PLC-β4 contained PLC activity. I also expressed chimeric PLC-β3/β4 and PLC-β4/β3 using the baculovirus system, and their expression was confirmed by the immunoblot method. However, chimeric PLC-β4/β3 did not show PLC activity, while chimeric PLC-β3/β4 retained its PLC-activity. Conclusion: Expression of chimeric PLC-β4 using the baculovirus system was an efficient method to obtain a large amount of protein. Moreover, this expression and purification method would be useful in studying the physical and chemical characteristics of this protein. In my study using chimeric PLC-β protein by swapping the N terminal and C terminal portions of PLC-β3 and β4, chimeric protein lost its activity completely in PLC-β4/β3 chimera. This result suggested a minute change in the tertiary structure of the protein, which may significantly affect its function.
Background: Although phospholipase C(PLC)-B3 is thought to be a very important enzyme in intracellular signal transduction, the sophisticated and complicated purification steps make it difficult to obtain sufficient amount of protein to study regulation of its activity by G proteins or other proteins. In order to get large amount of PLC-B3 protein, I employed baculovirus expression system which is known to express large amount of functionally active proteins. Methods: In order to make recombinant baculovirus which expresses PLC-B3 gene, partial cDNA of PLC-B3 which lacked 51 nucleotides was used to make full length PLC-B3 cDNA. By PCR, 5-end sequence of PLC-B3 was ligated into partial rat PLC-B3 cDNA and later cloned into pVL1393 transfer vector to make recombinant baculovirus. This recombinant baculovirus containing PLC-B3 sequence was used to infect Sf9 insect cells. Results: Infection of Sf9 cells with recombinant baculovirus rendered expression of 152 kDa-PLC-B3 protein, which was confirmed by immunoblot assay and PLC activity assay. Conclusion: The whole length PLC-B3 cDNA was expressed in Sf9 cells using baculovirus expression system. By using it, homogeneous enzyme is expected to be purified to study precise activation and regulation mechanisms of PLC-B3. (J Kor Soc Endocrinol 12:283-294, 1997)