Establishment of an In Vivo Report System for the Evaluation of Amber Suppression Activity in Escherichia coli

대장균에서 비천연 아미노산의 위치특이적 삽입을 위한 Amber Suppressor tRNA와 Aminoacyl-tRNA Synthetase의 Amber Suppression 활성측정시스템 개발

  • Kim, Kyung-Tae (Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies) ;
  • Park, Mi-Young (Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies) ;
  • Park, Jung-Chan (Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies)
  • 김경태 (한국외국대학교 자연과학대학 생명공학과) ;
  • 박미영 (한국외국대학교 자연과학대학 생명공학과) ;
  • 박중찬 (한국외국대학교 자연과학대학 생명공학과)
  • Received : 2009.05.27
  • Accepted : 2009.06.23
  • Published : 2009.06.30

Abstract

Site-specific incorporation of unnatural amino acids into proteins in vivo can be achieved by co-expression of an orthogonal pair of suppressor tRNA and engineered aminoacyl-tRNA synthetase (ARS) that specifically ligates an unnatural amino acid to the suppressor tRNA. As a step to establish this technique, here we generated an Escherichia coli reporter strain DH10B(Tn:lacZam) by integrating amber mutated lacZ gene into the chromosome of E. coli DH10B strain. In vivo expression of E. coli amber suppressor $tRNA^{Gln}$ produced blue colonies in culture plates containing X-Gal as well as dramatically increased $\beta$-galactosidase activity. In addition, expression of an orthogonal pair of Saccharomyces cerevisiae suppressor $tRNA^{Tyr}$ and tyrosyl-tRNA synthetase also produced blue colonies as well as moderate increase of $\beta$-galactosidase activity. These data demonstrate that our reporter strain will provide an efficient method to assess amber suppression in both qualitative and quantitative manners.

대장균에서 비천연 아미노산을 단백질 생합성시 특정 위치에 삽입하는 방법의 하나로 amber suppressor tRNA와 여기에 비천연 아미노산을 특이적으로 결합할 수 있는 변형된 aminoacyl-tRNA synthetase 쌍을 이용한다. 이러한 기술의 개발을 위해 필요한 여러 요소 중 하나는 이러한 시스템이 대장균에서 얼마나 잘 작동하는지를 확인할 수 있는 in vivo 보고시스템을 설정하는 것이다. 본 논문에서는 $\beta$-galactosidase 유전자의 N-말단에 amber 코돈을 삽입한 보고유전자를 제작하였으며, 이를 대장균(DH10B)의 chromosomal DNA에 삽입하여 DH10B(Tn:lacZam) 균주를 개발하였다. Genomic PCR과 Southern blot 분석을 통하여 lacZ amber 유전자가 대장균의 염색체에 삽입된 것을 확인하였으며, DH10B(Tn:lacZam)은 amber suppression을 유도할 수 있는 벡터가 형질 전환될 경우만 $\beta$-galactosidase 활성을 나타냈다. DH10B(Tn:lacZam)에 효모균의 amber suppressor $tRNA^{Tyr}$와 Tyrosyl-tRNA synthetase 쌍을 동시에 발현하는 벡터를 형질전환하였을 때, amber suppression에 의해서 $\beta$-galactosidase 활성이 나타났다. 하지만 이 활성은 대장균의 amber suppressor $tRNA^{Gln}$를 발현하는 pSupE2를 형질전환하였을 때와 비교 하여 매우 낮은 $\beta$-galactosidase 활성을 나타냈다. 이러한 결과는 DH10B(Tn:lacZam) 균주가 $\beta$-galactosidase 활성을 통하여 정성 및 정량적으로 in vivo amber suppression 활성을 비교 분석할 수 있는 특성을 가졌음을 나타낸다.

Keywords

References

  1. 김경태, 박중찬. 2001. In vivo amber suppression의 측정을 위한 reporter system의 개발. 한국외국어대학교 기초과학연구 11, 93-100
  2. Bain, J.D., C.G. Glabe, T.A. Dix, A.R. Chamberlin, and E.S. Diala. 1989. Biosynthetic site-specific incorporation of a non-natural amino acid into a polypeptide. J. Am. Chem. Soc. 111, 8013-8014 https://doi.org/10.1021/ja00202a052
  3. Bose, M., D. Groff, J. Xie, E. Brustad, and P.G. Schultz. 2006. The incorporation of a photoisomerizable amino acid into proteins in E. coli. J. Am. Chem. Soc. 128, 388-389 https://doi.org/10.1021/ja055467u
  4. Bradley, D., J.V. Park, and L. Soll. 1981. TRNA2Gln Su+2 mutants that increase amber suppression. J. Bacteriol. 145, 704-712
  5. De Lorenzo, V., M. Herrero, U. Jakubzik, and K.N. Timmis. 1990. Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. J. Bacteriol. 172, 6568-6572 https://doi.org/10.1128/jb.172.11.6568-6572.1990
  6. De Lorenzo, V., I. Cases, M. Herrero, and K.N. Timmis. 1993. Early and late responses of TOL promoters to pathway inducers: identification of postexponential promoters in Pseudomonas putida with lacZ-tet bicistronic reporters. J. Bacteriol. 175, 6902-6907 https://doi.org/10.1128/jb.175.21.6902-6907.1993
  7. Hohsaka, T., D. Kajihara, Y. Ashizuka, H. Murakami, and M. Sisido. 1999. Efficient incorporation of nonnatural amino acids with large aromatic groups into streptavidin in in vitro protein synthesizing systems. J. Am. Chem. Soc. 121, 34-40 https://doi.org/10.1021/ja9813109
  8. Kowal, A.K., C. Kohrer, and U.L. RajBhandary. 2001. Twentyfirst aminoacyl-tRNA synthetase suppressor tRNA pairs for possible use in site-specific incorporation of amino acid analogues into proteins in eukaryotes and in eubacteria. Proc. Natl. Acad. Sci. USA 98, 2268-2273 https://doi.org/10.1073/pnas.031488298
  9. Liu, D.R., T.J. Magliery, M. Pastrnak, and P.G. Schultz. 1997. Engineering a tRNA and aminoacyl-tRNA synthetase for the sitespecific incorporation of unnatural amino acids into proteins in vivo. Proc. Natl. Acad. Sci. USA 94, 10092-10097 https://doi.org/10.1073/pnas.94.19.10092
  10. Liu, D.R. and P.G. Schultz. 1999. Progress toward the evolution of an organism with an expended genetic code. Proc. Natl. Acad. Sci. USA 96, 4780-4785 https://doi.org/10.1073/pnas.96.9.4780
  11. Mendel, D., V.W. Cornish, and P.G. Schultz. 1995. Site-directed mutagenesis with an expanded genetic code. Annu. Rev. Biophys. Biomol. Struct. 24, 435-462 https://doi.org/10.1146/annurev.bb.24.060195.002251
  12. Noren, C.J., S.J. Anthony-Cahill, M.C. Griffith, and P.G. Schultz. 1989. A general method for site specific incorporation of unnatural amino acids into proteins. Science 244, 182-188 https://doi.org/10.1126/science.2649980
  13. Ohno, S., T. Yokogawa, I. Fujii, H. Asahara, H. Inokuchi, and K. Nishikawa. 1998. Co-expression of yeast amber suppressor tRNATyr and tyrosyl-tRNA synthetase in Escherichia coli: possibility to expand the genetic code. J. Biochem. 124, 1065-1068 https://doi.org/10.1093/oxfordjournals.jbchem.a022221
  14. Pastrnak, M., T.J. Magliery, and P.G. Schultz. 2000. A new orthogonal suppressor tRNA/aminoacyl-tRNA synthetase pair for evolving an organism with an expanded genetic code. Helv. Chim. Acta 83, 2277-2286 https://doi.org/10.1002/1522-2675(20000906)83:9<2277::AID-HLCA2277>3.0.CO;2-L
  15. Steer, B.A. and P. Schimmel. 1999. Major anticodon-binding region missing from an archaebacterial tRNA synthetase. J. Biol. Chem. 274, 35601-35606 https://doi.org/10.1074/jbc.274.50.35601
  16. Wang, L., A. Brock, B. Herberich, and P.G. Schultz. 2001. Expanding the genetic code of Escherichia coli. Science 292, 498-500 https://doi.org/10.1126/science.1060077
  17. Wang, L., J. Xie, and P.G. Schultz. 2006. Expanding the genetic code. Annu. Rev. Biophys. Biomol. Struct. 35, 225-249 https://doi.org/10.1146/annurev.biophys.35.101105.121507
  18. Xie, J., L. Wang, N. Wu, A. Brock, G. Spraggon, and P.G. Schultz. 2004. The site-specific incorporation of p-iodo-L-phenylalanine into proteins for structure determination. Nat. Biotechnol. 22, 1297-1301 https://doi.org/10.1038/nbt1013
  19. Zhang, Z., J. Gildersleeve, Y.Y. Yang, R. Xu, J.A. Loo, S. Uryu, C.H. Wong, and P.G. Schultz. 2004. A new strategy for the synthesis of glycoproteins. Science 303, 371-373 https://doi.org/10.1126/science.1089509