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Development of a Novel Cell Surface Attachment System to Display Multi-Protein Complex Using the Cohesin-Dockerin Binding Pair

  • Ko, Hyeok-Jin (Food Biotech R&D Center, Samyang Corp.) ;
  • Song, Heesang (Department of Biochemistry and Molecular Biology, Chosun University School of Medicine) ;
  • Choi, In-Geol (Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University)
  • Received : 2021.05.18
  • Accepted : 2021.07.05
  • Published : 2021.08.28

Abstract

Autodisplay of a multimeric protein complex on a cell surface is limited by intrinsic factors such as the types and orientations of anchor modules. Moreover, improper folding of proteins to be displayed often hinders functional cell surface display. While overcoming these drawbacks, we ultimately extended the applicability of the autodisplay platform to the display of a protein complex. We designed and constructed a cell surface attachment (CSA) system that uses a non-covalent protein-protein interaction. We employed the high-affinity interaction mediated by an orthogonal cohesin-dockerin (Coh-Doc) pair from Archaeoglobus fulgidus to build the CSA system. Then, we validated the orthogonal Coh-Doc binding by attaching a monomeric red fluorescent protein to the cell surface. In addition, we evaluated the functional anchoring of proteins fused with the Doc module to the autodisplayed Coh module on the surface of Escherichia coli. The designed CSA system was applied to create a functional attachment of dimeric α-neoagarobiose hydrolase to the surface of E. coli cells.

Keywords

Acknowledgement

This study was supported by a grant from Chosun University (2021).

References

  1. van Bloois E, Winter RT, Kolmar H, Fraaije MW. 2011. Decorating microbes: surface display of proteins on Escherichia coli. Trends Biotechnol. 29: 79-86. https://doi.org/10.1016/j.tibtech.2010.11.003
  2. Lee SY, Choi JH, Xu Z. 2003. Microbial cell-surface display. Trends Biotechnol. 21: 45-52. https://doi.org/10.1016/S0167-7799(02)00006-9
  3. Rutherford N, Mourez M. 2006. Surface display of proteins by gram-negative bacterial autotransporters. Microb. Cell Fact. 5: 22. https://doi.org/10.1186/1475-2859-5-22
  4. Nicolay T, Vanderleyden J, Spaepen S. 2015. Autotransporter-based cell surface display in Gram-negative bacteria. Crit. Rev. Microbiol. 41: 109-123. https://doi.org/10.3109/1040841X.2013.804032
  5. Jose J, Bernhardt R, Hannemann F. 2002. Cellular surface display of dimeric Adx and whole cell P450-mediated steroid synthesis on E. coli. J. Biotechnol. 95: 257-268. https://doi.org/10.1016/S0168-1656(02)00030-5
  6. Jose J, von Schwichow S. 2004. Autodisplay of active sorbitol dehydrogenase (SDH) yields a whole cell biocatalyst for the synthesis of rare sugars. Chembiochem. 5: 491-499. https://doi.org/10.1002/cbic.200300774
  7. Bielen A, Teparic R, Vujaklija D, Mrsa V. 2014. Microbial anchoring systems for cell-surface display of lipolytic enzymes. Food Technol. Biotechnol. 52: 16.
  8. Bayer EA, Belaich JP, Shoham Y, Lamed R. 2004. The cellulosomes: multienzyme machines for degradation of plant cell wall polysaccharides. Annu. Rev. Microbiol. 58: 521-554. https://doi.org/10.1146/annurev.micro.57.030502.091022
  9. Chauvaux S, Beguin P, Aubert JP, Bhat KM, Gow LA, Wood TM, et al. 1990. Calcium-binding affinity and calcium-enhanced activity of Clostridium thermocellum endoglucanase D. Biochem. J. 265: 261-265. https://doi.org/10.1042/bj2650261
  10. Choi SK, Ljungdahl LG. 1996. Structural role of calcium for the organization of the cellulosome of Clostridium thermocellum. Biochemistry 35: 4906-4910. https://doi.org/10.1021/bi9524631
  11. Fierobe HP, Mechaly A, Tardif C, Belaich A, Lamed R, Shoham Y, et al. 2001. Design and production of active cellulosome chimeras. Selective incorporation of dockerin-containing enzymes into defined functional complexes. J. Biol. Chem. 276: 21257-21261. https://doi.org/10.1074/jbc.M102082200
  12. Pages S, Belaich A, Fierobe HP, Tardif C, Gaudin C, Belaich JP. 1999. Sequence analysis of scaffolding protein CipC and ORFXp, a new cohesin-containing protein in Clostridium cellulolyticum: comparison of various cohesin domains and subcellular localization of ORFXp. J. Bacteriol. 181: 1801-1810. https://doi.org/10.1128/JB.181.6.1801-1810.1999
  13. Pages S, Belaich A, Tardif C, Reverbel-Leroy C, Gaudin C, Belaich JP. 1996. Interaction between the endoglucanase CelA and the scaffolding protein CipC of the Clostridium cellulolyticum cellulosome. J. Bacteriol. 178: 2279-2286. https://doi.org/10.1128/jb.178.8.2279-2286.1996
  14. Sakka K, Sugihara Y, Jindou S, Sakka M, Inagaki M, Sakka K, et al. 2011. Analysis of cohesin-dockerin interactions using mutant dockerin proteins. FEMS Microbiol. Lett. 314: 75-80. https://doi.org/10.1111/j.1574-6968.2010.02146.x
  15. Salama-Alber O, Jobby MK, Chitayat S, Smith SP, White BA, Shimon LJW, et al. 2013. Atypical cohesin-dockerin complex responsible for cell surface attachment of cellulosomal components: binding fidelity, promiscuity, and structural buttresses. J. Biol. Chem. 288: 16827-16838. https://doi.org/10.1074/jbc.M113.466672
  16. Voronov-Goldman M, Lamed R, Noach I, Borovok I, Kwiat M, Rosenheck S, et al. 2011. Noncellulosomal cohesin from the hyperthermophilic archaeon Archaeoglobus fulgidus. Proteins 79: 50-60. https://doi.org/10.1002/prot.22857
  17. Ko HJ, Park E, Song J, Yang TH, Lee HJ, Kim KH, et al. 2012. Functional cell surface display and controlled secretion of diverse Agarolytic enzymes by Escherichia coli with a novel ligation-independent cloning vector based on the autotransporter YfaL. Appl. Environ. Microbiol. 78: 3051-3058. https://doi.org/10.1128/AEM.07004-11
  18. Chung CT, Niemela SL, Miller RH. 1989. One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proc. Natl. Acad. Sci. USA 86: 2172-2175. https://doi.org/10.1073/pnas.86.7.2172
  19. Ekborg NA, Taylor LE, Longmire AG, Henrissat B, Weiner RM, Hutcheson SW. 2006. Genomic and proteomic analyses of the agarolytic system expressed by Saccharophagus degradans 2-40. Appl. Environ. Microbiol. 72: 3396-3405. https://doi.org/10.1128/AEM.72.5.3396-3405.2006
  20. Campbell RE, Tour O, Palmer AE, Steinbach PA, Baird GS, Zacharias DA, et al. 2002. A monomeric red fluorescent protein. Proc. Natl. Acad. Sci. USA 99: 7877-7882. https://doi.org/10.1073/pnas.082243699
  21. Ha SC, Lee S, Lee J, Kim HT, Ko HJ, Kim KH, et al. 2011. Crystal structure of a key enzyme in the agarolytic pathway, α-neoagarobiose hydrolase from Saccharophagus degradans 2-40. Biochem. Biophys. Res. Commun. 412: 238-244. https://doi.org/10.1016/j.bbrc.2011.07.073
  22. Nelson MD, Fitch DH. 2011. Overlap extension PCR: an efficient method for transgene construction. Methods Mol. Biol. 772: 459-470. https://doi.org/10.1007/978-1-61779-228-1_27
  23. Lee J, Kim SH. 2009. High-throughput T7 LIC vector for introducing C-terminal poly-histidine tags with variable lengths without extra sequences. Protein Expr. Purif. 63: 58-61. https://doi.org/10.1016/j.pep.2008.09.005
  24. Duckworth M, Yaphe W. 1970. Thin-layer chromatographic analysis of enzymic hydrolysates of agar. J. Chromatogr. 49: 482-487. https://doi.org/10.1016/S0021-9673(00)93663-X
  25. Schneider CA, Rasband WS, Eliceiri KW. 2012. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9: 671-675. https://doi.org/10.1038/nmeth.2089
  26. Haimovitz R, Barak Y, Morag E, Voronov-Goldman M, Shoham Y, Lamed R, et al. 2008. Cohesin-dockerin microarray: Diverse specificities between two complementary families of interacting protein modules. Proteomics 8: 968-979. https://doi.org/10.1002/pmic.200700486
  27. Arai R, Ueda H, Kitayama A, Kamiya N, Nagamune T. 2001. Design of the linkers which effectively separate domains of a bifunctional fusion protein. Protein Eng. 14: 529-532. https://doi.org/10.1093/protein/14.8.529
  28. Caspi J, Barak Y, Haimovitz R, Irwin D, Lamed R, Wilson DB, et al. 2009. Effect of linker length and dockerin position on conversion of a Thermobifida fusca endoglucanase to the cellulosomal mode. Appl. Environ. Microbiol. 75: 7335-7342. https://doi.org/10.1128/AEM.01241-09
  29. Wu CH, Mulchandani A, Chen W. 2008. Versatile microbial surface-display for environmental remediation and biofuels production. Trends Microbiol. 16: 181-188. https://doi.org/10.1016/j.tim.2008.01.003
  30. Leo JC, Grin I, Linke D. 2012. Type V secretion: mechanism(s) of autotransport through the bacterial outer membrane. Philos. Trans. R Soc. Lond B Biol. Sci. 367: 1088-1101. https://doi.org/10.1098/rstb.2011.0208
  31. Zarschler K, Janesch B, Kainz B, Ristl R, Messner P, Schaffer C. 2010. Cell surface display of chimeric glycoproteins via the S-layer of Paenibacillus alvei. Carbohydr. Res. 345: 1422-1431. https://doi.org/10.1016/j.carres.2010.04.010
  32. Liew PX, Wang CL, Wong SL. 2012. Functional characterization and localization of a Bacillus subtilis sortase and its substrate and use of this sortase system to covalently anchor a heterologous protein to the B. subtilis cell wall for surface display. J. Bacteriol. 194: 161-175. https://doi.org/10.1128/JB.05711-11
  33. Bello-Gil D, Maestro B, Fonseca J, Feliu JM, Climent V, Sanz JM. 2014. Specific and reversible immobilization of proteins tagged to the affinity polypeptide C-LytA on functionalized graphite electrodes. PLoS One 9: e87995. https://doi.org/10.1371/journal.pone.0087995
  34. Loimaranta V, Hytonen J, Pulliainen AT, Sharma A, Tenovuo J, Stromberg N, et al. 2009. Leucine-rich repeats of bacterial surface proteins serve as common pattern recognition motifs of human scavenger receptor gp340. J. Biol. Chem. 284: 18614-18623. https://doi.org/10.1074/jbc.M900581200
  35. Berlec A, Zadravec P, Jevnikar Z, Strukelj B. 2011. Identification of candidate carrier proteins for surface display on Lactococcus lactis by theoretical and experimental analyses of the surface proteome. Appl. Environ. Microbiol. 77: 1292-1300. https://doi.org/10.1128/AEM.02102-10
  36. Karpol A, Kantorovich L, Demishtein A, Barak Y, Morag E, Lamed R, et al. 2009. Engineering a reversible, high-affinity system for efficient protein purification based on the cohesin-dockerin interaction. J. Mol. Recognit. 22: 91-98. https://doi.org/10.1002/jmr.926