DOI QR코드

DOI QR Code

대장균에서 분자 chaperone에 의한 alginate lyase의 가용성 발현 증대

Enhancement of Soluble Expression of Alginate Lyase By Molecular Chaperone in E. coli.

  • 신은정 (동의대학교 바이오물질제어학과) ;
  • 이재형 (동의대학교 바이오물질제어학과) ;
  • 박소림 ((주)바이오리더스) ;
  • 김형락 (부경대학교 식품생명과학과) ;
  • 남수완 (동의대학교 바이오물질제어학과)
  • 발행 : 2007.01.29

초록

E. coli에서 Pseudoalteromonas elyakovii 유래의 alginate lyase유전자(aly)를 발현시킬 때, 대부분의 단백질이 불용성 내포체 형태로 발현됨을 확인하였다. Alginate lyase를 가용성 활성형으로 생산하기 위해 aly와 DnaK/DnaJ/GrpE 또는 aly와 GroEL/ES을 공발현하는 형질전환체를 얻었다. 공발현 결과, 단백질의 올바른 접힘을 도와주는 DnaK/DnaJ/GrpE chaperone이 가용성 및 활성형의 alginate lyase 생산에 매우 효과적임을 알 수 있었다. DnaK/DnaJ/GrpE chaperone의 발현에 유도제인 L-arabinose 최적 농도는 0.05 mg/ml이었으며, 이러한 공발현에 의해 약 34%의 alginate lyase가 가용성 분획에서 생산되었다. 또한 10%의 cetylpyridinium chloride를 첨가함으로써, 공발현 콜로니 주위에 투명환이 형성됨을 확인할 수 있었고, 이는 활성형 alginate lyase 효소에 의해 alginate가 분해되었음을 시사하였다.

When alginate lyase gene (aly) from Pseudoalteromonas elyakovii was expressed in E. coli, most of the gene product was produced as aggregated insoluble particles known as inclusion bodies. In order to produce a soluble and active form of alginate lyase, E. coli cells fore cotransformed with the plasmids designed to permit coexpression of aly together with molecular chaperones such as DnaK/DnaJ/GrpE or GroEL/ES chaperones. The results revealed that the coexpression of aly together with DnaK/DnaJ/GrpE chaperone had a marked effect on the production of this protein as a soluble and active form, presumably through facilitating correct folding of alginate lyase protein. The optimal concentration of L-arabinose for the induction of DnaK/DnaJ/GrpE chaperone was found to be 0.05 mg/ml. When DnaK/DnaJ/GrpE chaperone was coexpressed, about 34% in the total alginate lyase was produced in the soluble fraction. By addition of 10% cetylpyridinium chloride, a clear zone around the colony coexpressing aly and DnaK/DnaJ/GrpE chaperone was formed, indicating that the alginate in the medium was hydrolyzed by active alginate lyase enzyme.

키워드

참고문헌

  1. Baneyx, F., M. Mujacic. 2004. Recombinant protein folding and misfolding in Escherichia coli. Nat. Biotechnol. 22, 1399-1408 https://doi.org/10.1038/nbt1029
  2. Chen, Y., J. Song, S. F. Sui and D. N. Wang. 2003. DnaK and DnaJ facilitated the folding process and reduced inclusion body formation of magnesium transporter CorA overexpressed in Escherichia coli. Prot. Expr. Purif. 32, 221-231 https://doi.org/10.1016/S1046-5928(03)00233-X
  3. Gonzalez-Montalban, N., M. M. Carrio, S. Cuatrecasas, A. Aris and A. Villaverde. 2005. Bacterial inclusion bodies are cytotoxic in vivo in absence of functional chaperones DnaK or GroEL. J. Biotechnol. 10, 406-412
  4. Cragerov, A., E. Nudler, N. Komissarova, G. A. Gaitanaris, M. E. Gottesman and V. Nikiforov. 1992. Cooperation of GroEL/GroES and DnaK/DnaJ heat shock proteins in preventing protein misfolding in Escherichia coli. Proc. Natl. Acad. Sci. USA. 89, 10341-10344 https://doi.org/10.1073/pnas.89.21.10341
  5. Han, N. S. and B. Y. Tao. 1999. Enhancement of solubility of Bacillus macerans cyclodextrin glucanotransferase by thioredoxin fusion. Food Sci. Biotechnol. 8, 216-279
  6. Hartl, F. U. 1996. Molecular chaperones in cellular protein folding. Nature 381, 571-580 https://doi.org/10.1038/381571a0
  7. Kondo, A., J. Kohda, Y. Endo, T. Shiromizu, Y. Kurokawa, K. Nishihara, H. Yanagi, T. Yura and H. Fukuda. 2000. Improvement of productivity of active horseradish peroxidase in Escherichia coli by coexpression of Dsb proteins. J. Biosci. Bioeng. 90, 600-606 https://doi.org/10.1263/jbb.90.600
  8. Kwak, Y. H., S. J. Kim, K. Y. Lee and H. B. Kim. 2000. Stress responses of the Escherichia coli groE promoter. J. Microbiol. Biotechnol. 10, 63-68
  9. Kwon, M. J., S. L. Park, S. K. Kim and S. W. Nam. 2002. Overproduction of Bacillus macerans cyclodextrin glucanotransferase in E. coli by coexpression of GroEL/ES chaperone. J. Microbiol. Biotechnol. 12, 1002-1005
  10. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685 https://doi.org/10.1038/227680a0
  11. Mandel, M. and A. Higa. 1970. The calcium dependent bacteriophage DNA infection. J. Mol. Biol. 53, 159-162 https://doi.org/10.1016/0022-2836(70)90051-3
  12. Murata, K., T. Inose, T. Hisano, S. Abe, Y. Yonemoto, T. Yamashita, M. Takagi, K. Sakaguchi, A. Kimura and T. Imanaka. 1993. Bacterial alginate lyase: enzymoloy, genetics, and application. J. Ferment. Bioeng. 76, 427-437 https://doi.org/10.1016/0922-338X(93)90040-F
  13. Nishihar, K., M. Kanemori, H. Yanagi and T. Yura, 2000. Overexpression of trigger factor prevents aggregation of recombinant proteins in Escherichia coli. Appl. Environ. Microbiol. 66, 884-889 https://doi.org/10.1128/AEM.66.3.884-889.2000
  14. Nishihar, K., M. Kanemori, M. Kitagawa, H. Yanagi and T. Yura. 1998. Chaperone coexpression plasmids: differential and synergistic roles DnaK-DnaJ-GrpE and GroEL-GroES in assisting folding of an allergen of Japanese cedar pollen, Cryj2, in Escherichia coli. Appl. Environ. Microbiol. 64, 1694-1699
  15. Park, S. L., E. J. Shin, S. P. Hong, S. J. Jeon and S. W. Nam. 2005. Production of soluble human granulocyte colony stimulating factor in E. coli by molecular chaperones. J. Microbiol. Biotechnol. 15, 1267-1272
  16. Pecina, A., A. Pascual and A. Paneque. 1999. Cloning and expression of the algL gene, encoding the Azotobacter chroococcum alginate lyase: purification and characterization of the enzyme. J.Bacteriol. 181, 1409-1414
  17. Sachiko, M., Y. Yu, S. P. Singh, J. D. Kim, K Hayashi and Y. Kawata. 1998. Overproduction of $beta$-glucosidase in active form by an Escherichia coli system coexpressing the chaperonin GroEL/ES. FEBS Microbiol Lett. 159, 41-46
  18. Sawabe, T., H. Takahashi, Y. Ezura and P. Gacesa. 2001 Cloning, sequence analysis and expression of Pseudoalteromonas elyakoii IAM 14594 gene (alyPEEC) encoding the extracellular alginate lyase. Carbohydr. Res. 335, 11-21 https://doi.org/10.1016/S0008-6215(01)00198-7
  19. Standberg, L. and S. O. Enfors. 1991. Factors influencing inclusion body formation in the production of a fused protein in Escherichia coli. Appl. Environ. Microbiol. 57, 1669-1674
  20. Szabo, A., T. Langer, H. Schroder, J. Flanagan, B. Bukau and F. U. Hartl. 1994. The ATP hydrolysis-dependent reaction cycle of the Escherichia coli Hsp70 system-DnaK, DnaJ, and GrpE. Proc. Natl. Acad. Sci. USA. 91, 10345-10349 https://doi.org/10.1073/pnas.91.22.10345
  21. Thomas, J. G., A. Ayling and F. Baneyx. 1997. Molecular chaperones, folding catalysts, and the recovery of active recombinant proteins from E. coli. Appl. Biochem. Biotechnol. 66, 197-238 https://doi.org/10.1007/BF02785589
  22. Wall, J. G. and A. Pluckthun. 1995. Effects of overexpressing folding modulators on the in vivo folding of heterologous proteins in Escherichia coli. Curr. Opin. Biotechnol. 6, 507-516 https://doi.org/10.1016/0958-1669(95)80084-0
  23. Wegrzyn, R. D. and E. Deuerling. 2005. Molecular guardians for newborn proteins: ribosome-associated chaperones and their role in protein folding. Cell Mol. Life Sci. 62, 2727-2738 https://doi.org/10.1007/s00018-005-5292-z
  24. Wetzel, R. and B. A. Chrunyk. 1994. Inclusion body formation by interleukin-1 $\beta$ depends on the thermal sensitivity of a folding intermediate. FEBS Lett. 350, 245-248 https://doi.org/10.1016/0014-5793(94)00775-6
  25. Yoon, H. J. and W. Hashimoto, O. Miyake, M. Okamoto, B. Mikami, and K. Murata. 2000 Overexpression in Escherichia coli, purification and characterization of Sphingomonas sp. A1 alginate lyases. Prot. Expr. Purif. 19, 84-90 https://doi.org/10.1006/prep.2000.1226