Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Enhancement of Soluble Expression of CGTase in E. coli By Chaperone Molecules and Low Temperature Cultivation.

대장균에서 chaperons 분자와 저온배양에 의한 CGTase의 가용성 발현 증대

  • Published : 2004.02.01
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Abstract

The synergistic effect of lowered incubation temperature and CroEL/ES expression on the production of soluble form of B. macerans cyclodextrin glucanotransferase (CGTase) was studied in recombinant E. coli. pTCGTl and pGroll carrying the cgt and groEL/ES genes under the control of T7 promoter and pzt-I promoter, respectively, were co-introduced. Tetracycline (10 ng/ml) and IPTG (1 mM) were added at the early-exponential phase (2 hr) and mid-exponential phase (3 hr). Low temperature cultivation at $25^{\circ}C$ with groEL/ES expression improved the activity of CGTase by two fold, compared to $37^{\circ}C$ cultivation without chaperones. SDS-PACE analysis revealed that about 69% of CGTase in the total CGTase protein was found in the soluble fraction by overexpression of GroEL/ES and cultivation at$25^{\circ}C$, whereas 20% of CGTase was detected in the soluble fraction when E. coli was cultivated at $37^{\circ}C$ without chaperone. The amount of soluble CGTase from $25^{\circ}C$ culture with chaperone was 3.5-fold higher than that of $37^{\circ}C$ culture without chaperone. Therefore the expression of CroEL/ES and low temperature cultivation greatly enhanced the soluble production of CGTase in E. coli.

E. coli에서 B. macerans 유래 cyclodextrin glucanotransferase (CGTase)의 활성형 생산에 GroEL/ES chaperone과의 공발현과 저온 배양의 공동작용 효과에 대해 조사하였다. 실험에 사용된 cgt와 groEL/ES 유전자를 발현하는 pTCGTl과 pGroll은 각각 T7 promoter와 Pzt-1 promoter에 의해 조절되고 이들을 E. coli에 도입시켰다. 대수증식기 초기(2 hr)와 대수증식기 중기(3 hr)에 tetrarycline 10 ng/ml 과 IPTG 1 mM을 첨가하여 각각의 유전자를 발현시켰다. CGTase활성과 specific artivity 측정 시 $37^{\circ}C$에서 pTCGTl 단독 발현 보다 $25^{\circ}C$에서 chaperone과 함께 발현시킨 경우 2배나 높은 활성이 측정됐으며, SDS-PACE 분석결과 $37^{\circ}C$에서 단독 발현 시킬경우 20% 정도 가용성 형태로 발현되던 것이 $25^{\circ}C$에서 chaperone과 공발현 시에는 거의 3.5배가 넘는 69%가 가용성으로 전환됨을 알 수 있었다. 이와 같이 분자 chaperone과 $25^{\circ}C$에서의 저온 배양은 E. coli에서 활성형질 가용성 CGTase의 생산 증가에 큰 영향을 미치는 것으로 나타났다.

Keywords

References

  1. J. Biol. Chem. v.268 Inclusion body formation and protein stability in sequence variants of interleukin-1β Chrunyk,B.A.;J.Evans;J.Lillqust;P.Young;R.Wetzel
  2. Proteion Expression Purif. v.23 Chaperone-assisted overexpression of an active D-carbamolylase from Agrobacterium tumefaciens AM10 Dipti,S.;S.Rakesh;M.V.Rakesh https://doi.org/10.1006/prep.2001.1532
  3. Proc. Natl. Acad. Sci. USA v.89 Cooperation of GroEL/GroES and DnaK/DnaJ heat shock proteins in preventing protein misfolding in Escherichia coli Gragerov,A.;E.Nudler;N.Komissarova;G.A.Gaitanaris;M.E.Gottesman;V.Nikiforov https://doi.org/10.1073/pnas.89.21.10341
  4. Food Sci. Biotechnol. v.8 Enhancement of solubility of Bacillus macerans cyclodextrin glucanotransferase by thioredoxin fusion Han,N.S.;B.Y.Tao
  5. J. Microbiol. Biotechnol. v.11 Effects of environmental factors on n vivo folding of Bacillus macerans cyclodextrin glycosyltransferase in recombinant Escherichia coli Jin,H.H.;N.S.Han;D.K.Kweon;Y.C.Park;J.H.Seo
  6. J. Microbiol. Biotechnol. v.10 Refolding of Bacillus macerase cyclodextrin glucano-transferase expressed as inclusion bodies in recombinant Escherichia coli Kim,C.I.;M.D.Kim;Y.C.Park;N.S.Han;J.H.Seo
  7. Appl. Environ. Microbiol. v.61 Protein aggregation kinetics in an Escherichia coli strain overexpressing a Salmonella typhimurium CheY mutant gene Klein,J.;P.Dhurjati
  8. J. Biosci. Bioeng. v.90 Improvement of productivity of active horseradish peroxidase in Escherichia coli by coexpression of Dsb proteins Kondo,A.;J.Kohda;Y.Endo;T.Shiromizu;Y.Kurokawa;K.Nishihara;H.Yanagi;T.Yura;H.Fukuda https://doi.org/10.1263/jbb.90.600
  9. J. Microbiol. Biotechnol. v.12 Overproduction of Bacillus macerans cyclodextrin glucano-transferase in E. coli by coexpression of GroEL/ES chaperone Kwon,M.J.;S.L.Park;S.K.Kim;S.W.Nam
  10. Kor. J. Life Sci. v.12 Improvment of production of active cyclo-dextrin glucanotransferase by coexpression GroEL/ES chaperone in E. coil Kwon,M.J.;S.L.Park;B.W.Kim;S.K,Kim;S.W.Nam https://doi.org/10.5352/JLS.2002.12.6.688
  11. Starch v.46 High-level expression of cyclodextrin glucanotransferase in E. coli using a T7 promoter expression system Lee,P.K.C.;B.Y.Tao
  12. J. Biol. Chem. v.267 Effect of overproduction of heat shock chaperones GroESL and DnaK on human procollagenase production in Escherichia coli Lee,S.C.;P.O.Olins
  13. Anal. Biochem. v.181 A spectrophotometric assay for the cyclization activity of cyclomaltohexaose (a-cyclodextrin) glucanotransferase Lejeune,A.;K.Sakaguchi;T.Imanaka https://doi.org/10.1016/0003-2697(89)90385-0
  14. Appl. Environ. Microbiol. v.64 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 Nishihara,K.;M.Kanemori;M.Kitagawa;H.Yanagi;T.Yura
  15. Appl. Environ. Microbiol. v.66 Overexpression of trigger factor prevents aggregation of recombinant proteins in Escherichia coli Nishihara,K.;M.Kanemori;H.Yanagi;T.Yura https://doi.org/10.1128/AEM.66.3.884-889.2000
  16. Foods Biotechnol. v.4 Expression of cyclodextrin glucanotransferase from Bacillus macerans in recombinant Escherichia coli Park,Y.C.;C.S.Kim;N.S.Han;J.H.Seo
  17. J. Biol. Chem. v.263 Expression of soluble and fully functional ricin a chain in Escherichia coli is temperature sensitive Piatak,M.;J.A.Lane;W.Laird;M.J.Bjorn;A.Wang;M.Williams
  18. FEBS Microbiol Lett. v.159 Overproduction of β-glucosidase in active form by an Escherichia coli system coexpressing the chaperonin GroEL/ES Sachiko,M.;Y.Yu;S.P.Singh;J.D.Kim;K.Hayashi;Y.Kawata
  19. Appl. Environ. Microbiol. v.57 Factors influencing inclusion body formation in the production of a fused protein in Escherichia coli Standberg,L.;S.O.Enfors
  20. Proc. Natl. Acad. Sci. USA v.91 The ATP hydrolysis-dependent reaction cycle of the Escherichia coli Hsp70 system-Dnak, DnaJ, and GrpE Szabo,A.;T.Langer;H.Schroder;J.Flanagan;B.Bukau;F.U.Hartl https://doi.org/10.1073/pnas.91.22.10345
  21. Appl. Biochem. Biotechnol. v.66 Molecular chaperones, folding catalysts, and the recovery of active recombinant proteins from E. coli Thomas,J.G.;A.Ayling;F.Baneyx https://doi.org/10.1007/BF02785589
  22. Protein Expression Purif v.22 Expression of active hman C1 inhibitor serpin domain in E. coli Lamark,Y.;M.Ingebrigsten;C.Bjornstad;T.Melkko;T.E.Mollnes;E.W.Mielsen
  23. Curr. Opin. Biotechnol. v.6 Effects of over-expressing folding modulators on the in vivo folding of heterologous proteins in Escherichia coli Wall,J.G.;A.Pluckthun https://doi.org/10.1016/0958-1669(95)80084-0
  24. Cell v.83 Mechanism of GroEL action: productive release of polypeptide from a sequesterd position under GroES Weissman,J.S.;C.M.Hohl;O.Kovalenko;Y.Kashi;S.Chen;K.Braig;H.R.Saibil;W.A.Fenton;A.L.Horwich https://doi.org/10.1016/0092-8674(95)90098-5
  25. Cell v.84 Characterization of the active intermediate of a GroEL-GroES -mediated protein folding reaction Weissman,J.S.;H.S.Rye;W.A.Fenton;J.M.Beechem;A.L.Horwich https://doi.org/10.1016/S0092-8674(00)81293-3
  26. FEBS Lett. v.350 Inclusion body formation by interleukin-1β depends on the thermal sensitivity of a folding intermediate Wetzel,R.;B.A.Chrunyk https://doi.org/10.1016/0014-5793(94)00775-6
  27. J. Biol. Chem. v.268 Both the Escherichia coli chaperone systems, GroEL/GroES and DnaK/DnaJ/GrpE, can reactivate heat-treated RNA polymerase Ziemienowicz,A.;D.Skowyra;J. Zeilstra-Ryalls;O.Fayet;C.Georgopoulos;M.Zylicz