Applied Biological Chemistry

The Korean Society for Applied Biological Chemistry (한국응용생명화학회)
권호 표지

Overexpression and Purification of Bacillus subtilis Glutamyl-tRNA Synthetase in Escherichia coli

대장균에서 Bacillus subtilis glutamyl-tRNA synthetase의 과발현 및 정제

  • Oh, Jong-Shin (Department of Food Science and Technology, Dongguk University) ;
  • Yoon, Jang-Ho (Department of Food Science and Technology, Dongguk University) ;
  • Hong, Kwang-Won (Department of Food Science and Technology, Dongguk University)
  • Published : 2002.11.30
Download PDF
⟨ Previous Next ⟩

Abstract

Expression of Bacillus subtilis glutamyl-tRNA synthetase (GluRS) in Escherichia coli is lethal for the host, probably because this enzyme misaminoacylates ${tRNA_l}^{Gln}$ with glutamate in vivo. In order to overexpress B. subtilis GluRS, encoded by the gltX gene, in E. coli, this gene was amplified from B. subtilis 168 chromosomal DNA using PCR method and the entire coding region was cloned into a pET11a expression vector so that it was expressed under the control or the T7 Promoter. The resulting recombinant pEBER plasmid was transformed into E. coli Novablue (DE3) bearing the T7 RNA polymerase gene for expression. After IPTG treatment, the overproduced enzyme was purified using ammonium sulfate fractionation, Source Q anion exchange chromatography, Superdex-200 gel filtration, and Mono Q anion exchange chromatography. The purified enzyme yielded 18-fold increase in specific activity over the crude cell extract and its molecular weight was approximately 55 kDa on SDS-PAGE.

Bacillus subtilis의 glutamyl-tRNA synthetase(GluRS)는 대장균에서 발현될 때 숙주세포의 $tRNA_1^{Gln}$에 glutamate를 잘못 아실화하여 독성을 나타내는 것으로 추정되고 있다. 이러한 B. subtilis GluRS를 대장균에서 과발현 시키기 위하여 B. subtilis 168 균주의 chromosomal DNA에서 GluRS의 유전자(gltX)를 PCR을 이용하여 증폭하고 T7 promoter에 의해 발현이 조절되는 pET11a expression vector에 클로닝하였다. 이 재조합된 pEBER plasmid DNA로 T7 RNA polymerase를 갖는 대장균 NovaBlue(DE3)에 형질전환하였다. 형질전환된 대장균에 IPTG를 처리하여 과량 생성된 GluRS 단백질은 ammonium sulfate 분별침전 후 EPLC를 이용한 Source Q column anion exchange chromatography, Superdex 200 column gel filtration, Mono Q column anion exchange chromatography로 정제하였다. 정제된 B. subtilis의 GluRS 분자량은 약 55 kDa이었으며 효소의 활성도는 조효소액에 비해 18배로 증가하였다.

Keywords

References

  1. Yansura, D. G. and Henner, D. J. (1990) Use of the Escherichia coli trp promoter for direct expression of proteins. Methods Enzymol. 184, 54-60
  2. de Boer, H. A. and Hui, A. S. (1990) Sequences within ribosome binding site affecting messenger RNA translatability and method to direct hbosomes to single messenger RNA species. Methods Enzymol. 185, 103-114 https://doi.org/10.1016/0076-6879(90)85011-C
  3. Gerdes, K., Helin, K., Chhstensen, 0. W. and Lobner-Olesen, A. (1988) Protein translational control and differendal RNA decay are key elements regulating postsegregational expression of the killer protein encoded by the parB locus of plasmid Rl. J. Mol. Biol. 203, 119-29 https://doi.org/10.1016/0022-2836(88)90096-4
  4. Balbas, P. and Bolivar, F. (1990) Design and construction of expression plasmid vectors in Escherichia coti. Methods Enzymot. 185, 14-37 https://doi.org/10.1016/0076-6879(90)85005-9
  5. Studier, F. W., Rosenberg, A. H., Dunn, J. J. and Dubendorff, J. W. (1990) Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 185, 60-89 https://doi.org/10.1016/0076-6879(90)85008-C
  6. Proulx, M., Duplain, L., Lacoste, L., Yaguchi, M. and Lapointe, J. (1983) The monomeric glutamyl-tRNA synthetase from Bacillus subtilis 168 and its regulatory factor. J. Biol. Chem. 25, 753-759
  7. Lapointe, J., Duplain, L. and Proulx, M. (1986) A Single Glutamyl-tRNA Synthetase aminoacylates $tRNA^{Glu}$ and $tRNA^{Gln}$ in Bacittus subtitis and effciently misacylates Escherichia coti $tRNA^{Glu}$ in vitro. J. Bacteriol. 165, 88-93 https://doi.org/10.1128/jb.165.1.88-93.1986
  8. Pelchat, M., Lacoste, L., Yang, F. and Lapointe, J. (1998) Over- production of the Bacittus subtitis glutamyl-tRNA synthetase in its host and its toxicity to Escherichia coli. J. Microbiot. 44, 378-381
  9. Neubauer, R, Hofmann, K., Holst, 0., Mattiasson, B. and Kruschke, P. (1992) Maximizing the expression of a recombinant gene in Escherichia coli by manipulation of induction time using lactose as inducer. Appl Microbiol Biotechnol. 36, 739-744
  10. Beji, A., Izard, D., Gavini, F. and Leclerc, H. (1987) A rapid chemical procedure for isolation and purification of chromosomal DNA &om Gram-Negative Bacilli. Anatytical Biochem. 162, 18-23 https://doi.org/10.1016/0003-2697(87)90005-4
  11. Proulx, M. and Lapointe, J. (1985) Purification of Glutamyl-tRNA synthetase from Bacillus subtitis. Methods Enzvmol. 113, 50-54 https://doi.org/10.1016/S0076-6879(85)13010-7
  12. Lapoint, J., Levasseur, S. and Kem, D. (1985) Glutamyl-tRNA synthetase from Escherichia coli. Methods Enzymol. 3, 42-59
  13. Biisson, A. and Brun, Y. V. (1989) Overproduction and domain structure of the glutamyl-tRNA synthetase of Eschericttia coli. Biochem. Cell. Biol. 67, 404-410
  14. Breton, R., Watson, D. Yaguchi, M. and Lapointe, J. (1990) Glutamyl-tRNA synthetases of Bacillus subtitis 168T and of Baciltus steafvthennophitus. Cloning and sequencing of the gltX genes and comparison with other aminoacyl-tRNA synthetases. J. Biol. Chem. 265, 18248-18255
  15. Gendron, N., Breton, R., Champagne, N. and Lapointe, J. (1992) Adenylosuccinate lyase of Bacillus subtitis regulates the activity of the glutamyl-tRNA synfhetase. Proc. Natl. Acad. Sci. USA. 89, 5389-5392 https://doi.org/10.1073/pnas.89.12.5389
  16. Lapointe, J. and Soll, D. (1972) Glutamyl transfer ribonucleic acid synthetase of Escherichia coli. 3. Influence of the 46K protein on the affinity of the 56K glutamyl transfer ribonucleic acid synthetase for its substrates. J. Biol. Chem. 247, 4982-4985
  17. Powers, D. M. and Ginsburg, A. (1978) Monomeric structure of glutamyl-tRNA synthetase in Escherichia coli. Arch. Biochem. Biophys. 191, 673-679 https://doi.org/10.1016/0003-9861(78)90406-X