Bacillus subtilis의 단백질 분비기구 SecY의 유전자 수준의 조절이 단백질 분비에 미치는 영향

  • 김상숙 (명지대학교 생명과학과, 생명과학연구소) ;
  • 김순옥 (명지대학교 생명과학과, 생명과학연구소) ;
  • 서주원 (명지대학교 생명과학과, 생명과학연구소)
  • Published : 1996.08.01

Abstract

The SecY is a central component of the protein export machinery that mediate the translocation of secretory proteins across the plasma membrane, and has been known to be rate-limiting factor of secretion in Escherichia coli. In order to study the extracellular protein secretion in Gram-positive microorganism, we have, constructed strains harboring more than one copy of the gene for SecY. Firstly, the gene, for B. subtilis SecY and its promoter region was subcloned into pDH32 and the chimeric vector was inserted into amyE locus by homologous recombination. Secondly, low copy number vector, pCED6, was also used for subcloning the secY gene and for constructing a strain which harbors several copies of secY. The KH1 cell which harbor two copies of secY on the chromosome excreted more extracellular proteins than the wild type PB2. Moreover, the KH2 cells which harbor several copies of secY in pCED6 vector excreted more extracellular proteins than the KH1 cells. Here, we found that the capacity of protein secretion is partly controlled by the number of secY and it is suggested that SecY has also an important role in protein secretion in B. subtilis, a gram positive microorganism, as like in E. coli. This will promote the use of B. subtilis as a host for the expression of useful foreign gene and excretion of precious proteins.

Keywords

References

  1. Proc. Natl. Acad. Sci. USA. v.82 ATP is essential for protein translocation into Escherichia coli membrane vesicles Chen,L.;P.C.Tai
  2. EMBO. J. v.8 SeeA protein hydrolyzes ATP and is an essential component of the protein translocation APTase of Escherichia coli Lill,R.;K.Cunningham;L.A.Brundage;K.Ito;D.Oliver;W.Wickner
  3. J. Bacteriol. v.169 SecD, a new gene involved in protein export in Escherichia coli Gardel,S.C.;S.Benson;J.Hunt;S.Michaelis;J.Beckwith
  4. EMBO. J. v.9 The seeD locus of E. coli codes for two membrane proteins required for protein export Gardel,S.C.;K.Johnson;J.Beckwith
  5. Mol. Microbiol. v.6 SeeY and integral membrance components of the Escherichia coli protein translocation system Ito,K.
  6. Mol. Gen. Genet. v.197 Identification of the seeY gene product involved in protein export in Escherichia coli Ito,K.
  7. J. Bacteriol. v.160 Mutation that suppresses the protein export defect of the seeY mutation and causes cold-sensitive growth of Escherichia coli Shiba,K.;K.Ito.;T.Yura
  8. EMBO J. v.3 A defined mutation in the protein operon of Escherichia coli: Isolation and characterization of a new temperature-sensitive seeY mutant Shiba,K.;K.Ito.;T.Yura.;D.P.Cerretti
  9. EMBO J. v.6 Topology analysis of the SeeY protein, an integral membrance protein involved in protein export in Escherichia coli Akiyama,Y.;K.Ito
  10. Proc. Natl. Acad. Sci. USA. v.86 Site-specific antibodies against the PrlA (SeeY) protein of Escherichia coli inhibit protein export by interfering with plasma membrane binding of preprotein Watanabe,M.;G.Blobel
  11. Proc. Natl. Acad. Sci. USA. v.86 PrlA is important for the translocation of exported of protein across the cytoplasmic membrance of Escherichia coli Bieker.K.L.;T.J.Silhavy
  12. Molecular Microbiol. v.4 Isolation of a seeY homologue from Bacillus subtilis: Evidance for a common protein export pathway in eubacteria Suh,J.W.;S.A.Boylan;S.M.Thomas;K.M.Dolan;D.B.Oliver;C.W.Price
  13. Gene v.169 Genetic and transcriptional organization of the Bacillus subtilis spe-α region Suh,J.W.;S.A.Boylan;S.H.Oh;C.W.Price
  14. J. Bacteriol. v.114 Mapping of asporogenous mutations of Bacillus subtilis: a minimum estimate of the number of sporulation operons Piggot,P.J.
  15. Bacillus subtilis and other gram positive bacterial Integrational vectors for genetic manipulation in Bacillus subtilis Perego,M.;Sonenshein,A.L.(ed.);Hoch,J.L.(ed.);Losick.R.(ed.)
  16. J. Bacteriol. v.157 Promoter-probe plasmid for Bacillus subtilis Donnelly,C.E.;A.L.Sonenshein
  17. Molecular Cloning Sambrook,J.;E.F.Fritsch;T.Maniatis
  18. J. Bacteriol. v.153 Formation of competent Bacillus subtilis cells Sadaie,Y.;T.Kata
  19. Anal. Biochem. v.72 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the prociple of protein-dye binding Bradford,M.M.
  20. Anal. Biochem. v.86 Refinement of the coomassie blue method of protein quantitation Spector,T.
  21. Protein Method Bollag,D.M.;S.J.Edelstein