Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
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Construction, Expression, and Purification of N-Terminal Variants of Lumazine Protein from Photobacterium leiognathi

발광세균 Photobacterium leiognathi의 돌연변이 아미노-말단 루마진 단백질들의 제조, 발현 및 정제

  • Kang, Kyoung-Suk (Department of Biochemistry, Chungnam National University) ;
  • Kim, So-Young (Department of Biochemistry, Chungnam National University) ;
  • Choi, Ji-Sun (Department of Biochemistry, Chungnam National University) ;
  • Kim, Young-Doo (Department of Biochemistry, Chungnam National University) ;
  • Pokoo, Robert (Department of Biochemistry, Chungnam National University) ;
  • Nam, Ki-Seok (Department of Biochemistry, Chungnam National University) ;
  • Lee, Chan Yong (Department of Biochemistry, Chungnam National University)
  • Received : 2013.03.18
  • Accepted : 2013.06.24
  • Published : 2013.06.30
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Abstract

Lumazine protein is a fluorescent protein isolated from the bioluminescent bacteria of Photobacterium species. To generate minimal size of lumazine protein with possessing fluorescent characteristic, the gene coding for the wild type N-terminal domain of lumazine protein (N-LumP 118) containing amino acids up to 118 from Photobacterium leiognathi was produced. In addition, the genes coding for the variant proteins of N-LumP 118, replaced with one tryptophan amino acid (N-LumP 118 V41W, S48W, T50W, D64W, and A66W), were also constructed by Polymerase Chain Reaction and Site Directed Mutagenesis. These proteins were expressed in Escherichia coli by transformation with recombinant plasmids and purified by 6X-His tagging system. Spectroscopic studies have show that the purified proteins are capable of binding to the fluorescent ligand 6,7-dimethyl-8-ribityllumazine, resulted in showing of fluorescent characteristic with the minimal size of protein. From these studies, the mutant proteins containing single tryptophan amino acid residue, possessing its own intrinsic flouophore character at the different position, will be able to the use as a probe for further studies to deduce their three dimensional structure and the binding modes.

루마진 단백질은 발광 세균인 Photobacterium 종에서 추출된 형광성 단백질이다. 형광성을 지닌 최소 크기의 Photobacterium leiognathi 야생형 아미노-말단 도메인 루마진 단백질(N-terminal domain of lumazine protein 118 wt)과 여러 영역에 tryptophan을 생성시킨 돌연변이 단백질들(N-LumP 118 V41W, S48W, T50W, D64W, A66W)을 코드하는 유전자들을 위치 지정 돌연변이(Site Directed Mutagenesis)와 중합효소 연쇄 반응(Polymerase Chain Reaction)을 통해 제조하였다. 위의 유전자들이 포함된 재조합 플라스미드를 대장균에 형질 전환시켜 과발현시키는 최적의 조건을 찾았으며, 발현된 야생형 및 돌연변이 아미노-말단 영역 루마진 단백질을 6X-His tag system을 이용하여 정제 하였다. 흡광 및 형광 분광광도계를 이용한 실험 결과 이들 단백질들은 리간드인 6,7-dimethyl-8-ribityllumazine과 결합하여 형광성을 보유함을 보였다. 따라서 이들은 형광성을 지니게 되는 최소 크기의 루마진 단백질일 뿐만 아니라 형광성을 지닌 아미노산인 tryptophan이 여러 위치에 유일하게 존재함으로써 배향성 및 거리 등의 단백질의 구조 및 결합에 관한 심도 있는 연구에 탐침자로써 유용하게 활용 될 수 있을 것이다.

Keywords

References

  1. Campbell, A.K. 1989. Living light: biochemistry, function, and biomedical applications. Essays Biochem. 24, 41-76.
  2. Chatwell, L., Illarionova, V., Illarionov, B., Eisenreich, W., Huber, R., Skerra, A., Bacher, A., and Fischer, M. 2008. Structure of lumazine protein, an optical transponder of luminescent bacteria. J. Mol. Biol. 382, 44-55. https://doi.org/10.1016/j.jmb.2008.06.052
  3. Fischer, M. and Bacher, A. 2005. Biosynthesis of flavocoenzymes. Nat. Prod. Rep. 22, 324-350. https://doi.org/10.1039/b210142b
  4. Gast, R. and Lee, J. 1978. Isolation of in vivo emitter in bacterial bioluminescence. Proc. Natl. Acad. Sci. USA 75, 833-837. https://doi.org/10.1073/pnas.75.2.833
  5. Hastings, J.W. and Nealson, K.H. 1977. Bacterial bioluminescence. Annu. Rev. Microbiol. 31, 549-545. https://doi.org/10.1146/annurev.mi.31.100177.003001
  6. Herring, P.J. 1987. Systematic distribution of bioluminescence in living organisms. J. Biolumin. Chemilumin. 1, 147-163. https://doi.org/10.1002/bio.1170010303
  7. Illarionov, B., Eisenreich, W., Wirth, M., Lee,C.Y., Woo, Y.E., Bacher, A., and Fischer, M. 2007. Lumazine proteins from Photobacteria : localization of the single ligand binding site to the N-terminal domain. Biol. Chem. 388, 1313-1323.
  8. Illarionov, B., Illarionova, V., Lee, J., van Dongen, W., and Vervoort, J. 1994. Expression and properties of the recombination lumazine (riboflavin) protein from Photobacterium leiognathi. Biochim. Biophys. Acta 1201, 251-258. https://doi.org/10.1016/0304-4165(94)90048-5
  9. Koka, P. and Lee, J. 1979. Separation and structure of prosthetic group of the blue fluorescent protein from the bioluminescent bacterium Photobacterium phosphoreum. Proc. Natl. Acad Sci. USA 76, 3068-3072. https://doi.org/10.1073/pnas.76.7.3068
  10. Lee, C.Y., Illarionov, B., Woo, Y.E., Kemter, K., Kim, R.R., Eberhardt, S., Cushman, M., Eisenreich, W., Fischer, M., and Bacher, A. 2007. Ligand binding properties of the N-terminal domain of riboflavin synthase from Escherichia coli. J. Biochem. Mol. Biol. 40, 239-246. https://doi.org/10.5483/BMBRep.2007.40.2.239
  11. Lee, C.Y. and Meighen, E.A. 2000. The expression and DNA sequence of the gene coding for the lux specific fatty acyl-CoA reductase from Photobacterium phosphoreum. J. Microbiol. 38, 80-87.
  12. Lee, C.Y., O'Kane, D.J., and Meighen, E.A. 1994. Riboflavin synthesis genes are linked with the lux operon of Photobacterium phosphoreum. J. Bacteriol. 176, 2100-2104. https://doi.org/10.1128/jb.176.7.2100-2104.1994
  13. Meighen, E.A. 1988. Enzymes and genes from the lux operons of bioluminescent bacteria. Annu. Rev. Microbiol. 42, 151-179. https://doi.org/10.1146/annurev.mi.42.100188.001055
  14. Meighen, E.A. 1991. Molecular biology of bacterial bioluminescence. Microbiol. Rev. 55, 123-142.
  15. Meighen, E.A. 1994. Genetics of bacterial of bioluminescence. Annu. Rev. Genet. 28, 117-139. https://doi.org/10.1146/annurev.ge.28.120194.001001
  16. O'Kane, D.J., Karle, A.J., and Lee, J. 1985. Purification of lumazine protein from Photobacterium leiognathi and Photobacterium phosphoreum: bioluminescence properties. Biochemistry 24, 1454-1455.
  17. O'Kane, D.J. and Lee, J. 1985. Chemical characterization of lumazine protein from Photobacterium leiognathi: Comparison with lumazine protein from Photobacterium phosphoreum. Biochemistry 24, 1467-1475. https://doi.org/10.1021/bi00327a027
  18. O'Kane, D.J., Woodward, B., Lee, J., and Prasher, D.C. 1991. Borrowed proteins in bacterial bioluminescence. Proc. Natl. Acad. Sci. USA 88, 1100-1104. https://doi.org/10.1073/pnas.88.4.1100
  19. Palmer, I. and Wingfield, P.T. 1995. Preparation and extraction of insoluble (inclusion-body) proteins from Escherichia coli. pp. 6.3.1. -6.3.15. In Coligan, J.E., Dunn, B.M., Ploegh, H.L., Speicher, D.W., and Wingfield, P.T. (eds.). Current protocols in protein science, Vol. 1, John Wiley and Sons, New York, USA.
  20. Small, E.D., Koka, P., and Lee, J. 1980. Lmazine protein from the bioluminescent bacterium Photobacterium phosphoreum. Purification and characterization. J. Biol. Chem. 255, 8804-8810.