전체메뉴
검색
Article Search

JMB Journal of Microbiolog and Biotechnology

QR Code QR Code

Related articles in JMB

More Related Articles

Article

J. Microbiol. Biotechnol. 2006; 16(10): 1583-1590

Published online October 28, 2006

Copyright © The Korean Society for Microbiology and Biotechnology.

High-Level Expression of T4 Endonuclease V in Insect Cells as Biologically Active Form

Kang, Chang Soo , Seung-Yeol Son 1 and In Seok Bang 2*

Department of Biological Science, Hoseo University, Asan 336-795, Korea, 1Department of Microbiology and Institute of Basic Science, Dankook University, Cheonan 330-714, Korea, 2MyGene Bioscience Institute, Seoul 135-826, Korea

Abstract

T4 endonuclease V (T4 endo V) [EC 3. 1. 25. 1], found in bacteriophage T4, is responsible for excision repair of damaged DNA. The enzyme possesses two activities: a cyclobutane pyrimidine dimer DNA glycosylase (CPD glycosylase) and an apyrimidic/apurinic endonuclease (AP lyase). T4 denV (414 bp cDNA) encoding T4 en do V (138 amino acid) was synthesized and expressed using either an expression vector, pTriEx-4, in E. coli or a baculovirus AcNPV vector, pBacPAK8, in insect cells. The recombinant His-Tag/T4 endo V (rHis-Tag/T4 endo V) protein expressed from bacteria was purified using one-step affinity chromatography with a HiTrap Chelating HP column and used to make rabbit anti-His-Tag/T4 endo V polyclonal antibody for detection of recombinant T4 endo V (rT4 endo V) expressed in insect cells. In the meantime, the recombinant baculovirus was obtained by cotransfection of BacPAK6 viral DNA and pBP/T4 endo V in Spodoptera frugiperda (Sf21) insect cells, and used to infect Sf21 cells to overexpress T4 endo V protein. The level of rT4 endo V protein expressed in Sf21 cells was optimized by varying the virus titers and time course of infection. The optimal expression condition was set as follows; infection of the cells at a MOI of 10 and harvest at 96 h post-infection. Under these conditions, we estimated the amount of rT4 endo V produced in the baculovirus expression vector system to be 125 mg/l. The rT4 endo V was purified to homogeneity by a rapid procedure, consisting of ion-exchange, affinity, and reversed phase chromatographies, based on FPLC. The rT4 endo V positively reacted to an antiserum made against rHis-Tag/T4 endo V and showed a residual nicking activity against CPD-containing DNA caused by UV. This is the first report to have T4 endo V expressed in an insect system to exclude the toxic effect of a bacterial expression system, retaining enzymatic activity.

Keywords: AP lyase, baculovirus expression, bacteriophage T4, T4 endonuclease V, CPD glycosylase