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논문 기본 정보
- 자료유형
- 학술저널
- 저자정보
- 저널정보
- 한국미생물생명공학회 Journal of Microbiology and Biotechnology Journal of Microbiology and Biotechnology 제16권 제10호
- 발행연도
- 2006.1
- 수록면
- 1,583 - 1,590 (8page)
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초록· 키워드
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T4 endonuclease V (T4 endo V) [EC 3. 1. 25. 1],found in bacteriophage T4, is responsible for excision repairof damaged DNA. The enzyme possesses two activities: acyclobutane pyrimidine dimer DNA glycosylase (CPDglycosylase) and an apyrimidic/apurinic endonuclease (APlyase). T4 denV (414 bp cDNA) encoding T4 endo V (138expresion vector, pTriEx-4, in E. coli or a baculovirus AcNPVvector, pBacPAK8, insect cels. The recombinant His-Tag/T4 endo V (rHis-Tag/T4 endo V) protein expressed frombacteria was purified using one-step affinity chromatographywith a HiTrap Chelating HP column and used to make rabitanti-His-Tag/T4 endo V polyclonal antibody for detection ofrecombinant T4 endo V (rT4 endo V) expressed in insect cells.In the meantime, the recombinant baculovirus was obtainedV in Spodoptera frugiperda (Sf21) insect cells, and used toinfect Sf21 cells to overexpres T4 endo V protein. The levelof rT4 endo V protein expressed in Sf21 cells was optimizedby varying the virus titers and time course of infection. Theoptimal expresion condition was set as follows; infection ofthe cells at a MOI of 10 and harvest at 96 h post-infection.Under these conditions, we estimated the amount of rT4 endoV produced in the baculovirus expresion vector system to berapid procedure, consisting of ion-exchange, affinity, and reversed-phase chromatographies, based on FPLC. The rT4 endo Vpositively reacted to an antiserum made against rHis-Tag/T4endo V and showed a residual nicking activity against CPD-containing DNA caused by UV. This is the first report to have T4endo V expressed in an insect system to exclude the toxic effectof a bacterial expresion system, retaining enzymatic activity.
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참고문헌 (25)
참고문헌 신청
Altmann, F / 1999 / Insect cells as hosts for the expression of recombinant glycoproteins / Glycoconjugate J. 16 : 109 ~ 123
Bahia, D / 2005 / Optimisation of insect cell growth in deep-well blocks: Development of a high-throughput insect cell expression screen / Protein Express. Purif. 39 : 61 ~ 7
Bradford, M. M / 1976 / A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding / Anal. Biochem. 7
Chen, Yin / 2005 / Overexpression and Characterizaion of appA Phytase Expressed by Baculovirus-infected Silkworm / Journal of Microbiology and Biotechnology 15 (3) : 466 ~ 47
Dodson, M. L / 1993 / Evidence for an imino intermediate in the T4 endonuclease V reaction / Biochemistry 32 : 8284 ~ 8290
Bahia, D / 2005 / Optimisation of insect cell growth in deep-well blocks: Development of a high-throughput insect cell expression screen / Protein Express. Purif. 39 : 61 ~ 7
Bradford, M. M / 1976 / A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding / Anal. Biochem. 7
Chen, Yin / 2005 / Overexpression and Characterizaion of appA Phytase Expressed by Baculovirus-infected Silkworm / Journal of Microbiology and Biotechnology 15 (3) : 466 ~ 47
Dodson, M. L / 1993 / Evidence for an imino intermediate in the T4 endonuclease V reaction / Biochemistry 32 : 8284 ~ 8290
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