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The Korean Society for Biotechnology and Bioengineering (한국생물공학회)
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Screening of Bacterial Surface Display Anchoring Motif Using Tetrameric β-galactosidase in Bacillus subtilis Spore

Tetrameric β를 이용한 고초균 포자에서의 미생물 표면 발현 모체 선별

  • Kim, June-Hyung (Department of Chemical Engineering, Dong-A University) ;
  • Pan, Jae-Gu (Systems Microbiology Research Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Kim, Byung-Gee (School of Chemical Engineering, Seoul National University)
  • Received : 2011.05.17
  • Accepted : 2011.06.03
  • Published : 2011.06.30
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Abstract

Using tetrameric ${\beta}$-galactosidase as a model protein, anchoring motives were screened in Bacillus subtilis spore display system. Eleven spore coat proteins were selected considering their expression levels and the location in the spore coat layer. After chromosomal single-copy homologous integration in the amyE site of Bacillus subtilis chromosome, cotE and cotG were chosen as possible spore surface anchoring motives with their higher whole cell ${\beta}$-galactosidase activity. PAGE and Wester blot of extracted fraction of outer layer of purified spore, which express CotE-LacZ or CotG-LacZ fusion verified the existence of exact size of fusion protein and its location in outer coat layer of purified spore. ${\beta}$-galactosidase activity of spore with CotE-LacZ or CotG-LacZ fusion reached its highest value around 16~20 h of culture time in terms of whole cell and purified spore. After intensive spore purification with lysozyme treatment and renografin treatment, spore of BJH135, which expresses CotE-LacZ, retained only 1~2% of its whole cell ${\beta}$-galactosidase activity. Whereas spore of BJH136, which has cotG-lacZ cassette in the chromosome, retained 10~15% of its whole cell ${\beta}$-galactosidase activity, proving minor perturbation of CotG-LacZ, when incorporated in the spore coat layer of Bacillus subtilis compared to CotE-LacZ. Usage of Bacillus subtilis WB700, of which 7 proteases are knocked-out and thereby resulting in 99.7% decrease in protease activity of the host, did not prevent the proteolytic degradation of spore surface expressed CotG-LacZ fusion protein.

Keywords

References

  1. Charbit, A., A. Molla, W. Saurin, and M. Hofnung (1988) Versatility of a vector for expressing foreign polypeptides at the surface of gram-negative bacteria. Gene 70: 181-189. https://doi.org/10.1016/0378-1119(88)90116-3
  2. Boder, E. T. and K. D. Wittrup (1997) Yeast surface display for screening combinatorial polypeptide libraries. Nat. Biotechnol. 15: 553-557. https://doi.org/10.1038/nbt0697-553
  3. Jung, H. C., J. M. Lebeault, and J. G. Pan (1998) Surface display of Zymomonas mobilis levansucrase by using the ice-nucleation protein of Pseudomonas syringae. Nat. Biotechnol. 16: 576-580. https://doi.org/10.1038/nbt0698-576
  4. Georgiou, G., C. Stathopoulos, P. S. Daugherty, A. R. Nayak, B. L. Iverson, and R. Curtiss (1997) Display of heterologous proteins on the surface of microorganisms: from the screening of combinatorial libraries to live recombinant vaccines. Nat. Biotechnol. 15: 29-34. https://doi.org/10.1038/nbt0197-29
  5. Richins, R. D., I. Kaneva, A. Mulchandani, and W. Chen (1997) Biodegradation of organophosphorus pesticides by surfaceexpressed organophosphorus hydrolase. Nat. Biotechnol. 15: 984-987. https://doi.org/10.1038/nbt1097-984
  6. Sousa, C., A. Cebolla, and V. de Lorenzo (1996) Enhanced metalloadsorption of bacterial cells displaying poly-His peptides. Nat. Biotechnol. 14: 1017-1020. https://doi.org/10.1038/nbt0896-1017
  7. Agterberg, M., H. Adriaanse, A. van Bruggen, M. Karperien, and J. Tommassen (1993) Outer-membrane PhoE protein of Escherichia coli K -12 as an exposure vector: possibilities and limitations. Gene 88: 37-45.
  8. Dhillon, J. K., P. D. Drew, and A. J. Porter (1999) Bacterial surface display of an anti-pollutant antibody fragment. Lett. Appl. Microbiol. 28: 350-354. https://doi.org/10.1046/j.1365-2672.1999.00552.x
  9. Hansson, M., S. Stahl, T. N. Nguyen, T. Bachi, A. Robert, H. Binz, A. Sjolander, and M. Uhlen (1992) Expression of recombinant proteins on the surface ofthe coagulase-negative bacterium Staphylococcus xylosus. J. Bacteriol. 174: 4239-4245.
  10. Pozzi, G., M. Contorni, M. R. Oggioni, R. Manganelli, M. Tommasino, F. Cavalieri, and V. A. Fischetti (1992) Delivery and expression of a heterologous antigen on the surface of Streptococci. Infect. Immun. 60: 1902-1907 .
  11. Georgiou, G., D. L. Stephens, C. Stathopoulos, H. L. Poetschke, J. Mendenhall, and C. F. Earhart (1996) Display of beta-Iactamase on the Escherichia coli surface: outer membrane phenotypes conferred by Lpp'-OmpA'-beta-lactamase fusions. Protein Eng. 9: 239-247. https://doi.org/10.1093/protein/9.2.239
  12. Stathopoulos, C., G. Georgiou, and C. F. Earhart (1996) Characterization of Escherichia coli expressing an Lpp'OmpA (46-159)-PhoA fusion protein localized in the outer membrane. Appl. Microbiol. Biotechnol. 45: 112-119. https://doi.org/10.1007/s002530050657
  13. Steidler, L., E. Remaut, and W. Fiers (1993) LamB as a carrier molecule for the functional exposition ofIgG-binding domains of the Staphylococcus aureus protein A at the surface of Escherichia coli K12. Mol. Gen. Genet. 236: 87-192.
  14. Kornacker, M. G. and A. P. Pugsley (1990) The normally periplasmic enzyme beta-lactamase is specifically and efficiently translocated through the Escherichia coli outer membrane when it is fused to the cell-surface enzyme pullulanase. Mol. Microbiol. 4: 1101-1109. https://doi.org/10.1111/j.1365-2958.1990.tb00684.x
  15. Kaneshiro, C. M., C. A. Enns, M. G. Hahn, J. S. Peterson, and F. J. Reithel (1975) Evidence for an active dimer of Escherichia coli beta-galactosidase. Biochem. J. 151: 433-434.
  16. Harwood, C. R. and S. M. Cutting (1990) Molecular Biological Methods for Bacillus. pp. 67. A Wiley Interscience Publication, John Wiley & Sons Ltd., Chichester, West Sussex, England.
  17. Henriques, A. O., L. R. Melsen, and C. P. Moran Jr. (1998) Involvement of superoxide dismutase in spore coat assembly in Bacillus subtilis. J. Bacteriol. 180: 2285-2291.
  18. Zheng, L. and R. Losick (1990) Cascade regulation of spore coat gene expression in Bacillus subtilis. J Mol. BioI. 212: 645-660. https://doi.org/10.1016/0022-2836(90)90227-D
  19. Sacco, M., E. Ricca, R. Losick, and S. Cutting (1995) An additional GerE-controlled gene encoding an abundant spore coat protein from Bacillus subtilis. J. Bacteriol. 177: 372-377.
  20. Kim, J. H. and B. G. Kim (2000) Application of LFH-PCR for the Disruption of SpoIIIE and SpoIIIG of B. subtilis. Biotechnol. Bioprocess Eng. 5: 327-331. https://doi.org/10.1007/BF02942207
  21. Zheng, L., W. P. Donovan, P. C. Fitz-James, and R. Losick (1988) Gene encoding a morphogenic protein required in the assembly of the outer coat of the Bacillus subtilis endospore. Genes Dev. 2: 1047-1054. https://doi.org/10.1101/gad.2.8.1047
  22. Kim, J. and W. Schumann (2009) Display of proteins on Bacillus subtilis endospores. Cell. Mol. Life. Sci. 66: 3127-3136. https://doi.org/10.1007/s00018-009-0067-6