Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지
DOI QR코드

DOI QR Code

Construction, Investigation and Application of TEV Protease Variants with Improved Oxidative Stability

  • Bayar, Enkhtuya (School of Life Science, Anhui Agricultural University) ;
  • Ren, Yuanyuan (School of Life Science, Anhui Agricultural University) ;
  • Chen, Yinghua (School of Life Science, Anhui Agricultural University) ;
  • Hu, Yafang (School of Life Science, Anhui Agricultural University) ;
  • Zhang, Shuncheng (School of Life Science, Anhui Agricultural University) ;
  • Yu, Xuelian (School of Life Science, Anhui Agricultural University) ;
  • Fan, Jun (School of Life Science, Anhui Agricultural University)
  • Received : 2021.06.25
  • Accepted : 2021.09.08
  • Published : 2021.12.28
Download PDF
⟨ Previous Next ⟩

Abstract

Tobacco etch virus protease (TEVp) is a useful tool for removing fusion tags, but wild-type TEVp is less stable under oxidized redox state. In this work, we introduced and combined C19S, C110S and C130S into TEVp variants containing T17S, L56V, N68D, I77V and S135G to improve protein solubility, and S219V to inhibit self-proteolysis. The solubility and cleavage activity of the constructed variants in Escherichia coli strains including BL21(DE3), BL21(DE3)pLys, Rossetta(DE3) and Origami(DE3) under the same induction conditions were analyzed and compared. The desirable soluble amounts, activity, and oxidative stability were identified to be reluctantly favored in the TEVp. Unlike C19S, C110S and C130S hardly impacted on decreasing protein solubility in the BL21(DE3), but they contributed to improved tolerance to the oxidative redox state in vivo and in vitro. After two fusion proteins were cleaved by purified TEVp protein containing double mutations under the oxidized redox state, the refolded disulfide-rich bovine enterokinase catalytic domain or maize peroxidase with enhanced yields were released from the regenerated amorphous cellulose via affinity absorption of the cellulose-binding module as the affinity tag.

Keywords

Acknowledgement

This study was supported by the Anhui Educational Committee Foundation (KJ2020A0113).

References

  1. Yadav D K, Yadav N, Yadav S, Haque S, Tuteja N. 2016. An insight into fusion technology aiding efficient recombinant protein production for functional proteomics. Arch. Biochem. Biophys. 612: 57-77. https://doi.org/10.1016/j.abb.2016.10.012
  2. Cesaratto F, Burrone OR, Petris G. 2016. Tobacco Etch Virus protease: a shortcut across biotechnologies. J. Biotechnol. 231: 239-249. https://doi.org/10.1016/j.jbiotec.2016.06.012
  3. Cabrita LD, Gilis D, Robertson AL, Dehouck Y, Rooman M, Bottomley SP. 2007. Enhancing the stability and solubility of TEV protease using in silico design. Protein Sci. 16: 2360-2367. https://doi.org/10.1110/ps.072822507
  4. van den Berg S, Lofdahl PA, Hard T, Berglund H. 2006. Improved solubility of TEV protease by directed evolution. J. Biotechnol. 121: 291-298. https://doi.org/10.1016/j.jbiotec.2005.08.006
  5. Kapust RB, Tozser J, Fox JD, Anderson DE, Cherry S, Copeland TD, 2001. Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. Protein Eng. 14: 993-1000. https://doi.org/10.1093/protein/14.12.993
  6. Cesaratto F, Lopez-Requena A, Burrone OR, Petris G. 2015. Engineered tobacco etch virus (TEV) protease active in the secretory pathway of mammalian cells. J. Biotechnol. 212: 159-166. https://doi.org/10.1016/j.jbiotec.2015.08.026
  7. Phan J, Zdanov A, Evdokimov AG. Tropea JE, Peters HK. Kapust RB, et al. 2002. Structural basis for the substrate specificity of tobacco etch virus protease. J. Biol. Chem. 277: 50564-50572. https://doi.org/10.1074/jbc.M207224200
  8. Nunn CM, Jeeves M, Cliff MJ, Urquhart GT, George RR, Chao LH, et al. 2005. Crystal structure of tobacco etch virus protease shows the protein C terminus bound within the active site. J. Mol. Biol. 350: 145-155. https://doi.org/10.1016/j.jmb.2005.04.013
  9. Baeshen MN, Al-Hejin AM, Bora RS, Ahmed MM, Ramadan HA, Saini KS, et al. 2015. Production of biopharmaceuticals in E. coli: current scenario and future perspectives. J. Microbiol. Biotechnol. 25: 953-962. https://doi.org/10.4014/jmb.1412.12079
  10. Studier FW. 1991. Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system. J. Mol. Biol. 219: 37-44. https://doi.org/10.1016/0022-2836(91)90855-z
  11. Salinas G, Pellizza L, Margenat M, Flo M, Fernandez C. 2011. Tuned Escherichia coli as a host for the expression of disulfide-rich proteins. Biotechnol. J. 6: 686-699. https://doi.org/10.1002/biot.201000335
  12. Fang J, Chen L, Cheng B, Fan J. 2013. Engineering soluble tobacco etch virus protease accompanies the loss of stability. Protein Expr. Purif. 92: 29-35. https://doi.org/10.1016/j.pep.2013.08.015
  13. Zhou C, Yan Y, Fang J, Cheng B, Fan, J. 2014. A new fusion protein platform for quantitatively measuring activity of multiple proteases. Microb. Cell Fact. 13: 44. https://doi.org/10.1186/1475-2859-13-44
  14. Hwang PM, Pan JS, Sykes BD. 2014. Targeted expression, purification, and cleavage of fusion proteins from inclusion bodies in Escherichia coli. FEBS Lett. 588: 247-252. https://doi.org/10.1016/j.febslet.2013.09.028
  15. Jungbauer A, Kaar W, Schlegl R. 2004. Folding and refolding of proteins in chromatographic beds. Curr. Opin. Biotechnol. 15: 487-494. https://doi.org/10.1016/j.copbio.2004.08.009
  16. Berdichevsky Y, Lamed R, Frenkel D, Gophna U, Bayer EA, Yaron S, et al. 1999. Matrix-assisted refolding of single-chain Fv-cellulose binding domain fusion proteins. Protein Expr. Purif. 17: 249-259. https://doi.org/10.1006/prep.1999.1125
  17. Hong J, Wang Y, Ye X, Zhang YH. 2008. Simple protein purification through affinity adsorption on regenerated amorphous cellulose followed by intein self-cleavage. J. Chromatogr. A, 1194:150-154. https://doi.org/10.1016/j.chroma.2008.04.048
  18. Wingfield PT, Palmer I, Liang SM. 2014. Folding and purification of insoluble (inclusion body) proteins from Escherichia coli. Curr. Protoc. Protein Sci. 78: 6.5.1-6.5.30.
  19. Asial I, Cheng YX, Engman H, Dollhopf M, Wu B, Nordlund P, et al. 2013. Engineering protein thermostability using a generic activity-independent biophysical screen inside the cell. Nat. Commun. 4: 2901. https://doi.org/10.1038/ncomms3901
  20. Sanchez MI, Ting AY. 2020. Directed evolution improves the catalytic efficiency of TEV protease. Nat. Methods 17:167-174. https://doi.org/10.1038/s41592-019-0665-7
  21. Denard CA, Paresi C, Yaghi R, McGinnis N, Bennett Z, Yi L, et al. 2021. YESS 2.0, a tunable platform for enzyme evolution, yields highly active TEV protease variants. ACS Synth. Biol. 10: 63-71. https://doi.org/10.1021/acssynbio.0c00452
  22. Tan H, Wang J, Zhao ZK. 2007. Purification and refolding optimization of recombinant bovine enterokinase light chain overexpressed in Escherichia coli. Protein Expr. Purif. 56: 40-47. https://doi.org/10.1016/j.pep.2007.07.006
  23. Yu X, Sun J, Wang W, Jiang L, Wang R, Xiao W, et al. 2017. Tobacco etch virus protease mediating cleavage of the cellulose-binding module tagged colored proteins immobilized on the regenerated amorphous cellulose. Bioprocess Biosyst. Eng. 40: 1101-1110. https://doi.org/10.1007/s00449-017-1772-4
  24. Levy I, Ward G, Hadar Y, Shoseyov O, Dosoretz CG. 2003. Oxidation of 4-bromophenol by the recombinant fused protein cellulose-binding domain-horseradish peroxidase immobilized on cellulose. Biotechnol. Bioeng. 82: 223-231. https://doi.org/10.1002/bit.10562
  25. Skala W, Goettig P, Brandstetter H. 2013. Do-it-yourself histidine-tagged bovine enterokinase: a handy member of the protein engineer's toolbox. J. Biotechnol. 168: 421-425. https://doi.org/10.1016/j.jbiotec.2013.10.022
  26. Tao YM, Wang S, Luo HL, Yan WW. 2018. Peroxidase from jackfruit: Purification, characterization and thermal inactivation. Int. J. Biol. Macromol. 114: 898-905. https://doi.org/10.1016/j.ijbiomac.2018.04.007
  27. Fang J, Zou L, Zhou X, Cheng B, Fan J. 2014. Synonymous rare arginine codons and tRNA abundance affect protein production and quality of TEV protease variant. PLoS One 9: e112254. https://doi.org/10.1371/journal.pone.0112254
  28. Gupta RD. Tawfik DS. 2008. Directed enzyme evolution via small and effective neutral drift libraries. Nat. Methods 5: 939-942. https://doi.org/10.1038/nmeth.1262
  29. Yurkova MS, Sharapova OA, Zenin VA, Fedorov AN. 2019. Versatile format of minichaperone-based protein fusion system. Sci. Rep. 9: 15063. https://doi.org/10.1038/s41598-019-51015-0
  30. Kapust RB, Waugh DS. 2000. Controlled intracellular processing of fusion proteins by TEV protease. Protein Expr. Purif. 19: 312-318. https://doi.org/10.1006/prep.2000.1251
  31. Zhang W, Zheng W, Mao M, Yang Y. 2014. Highly efficient folding of multi-disulfide proteins in superoxidizing Escherichia coli cytoplasm. Biotechnol. Bioeng. 111: 2520-2527. https://doi.org/10.1002/bit.25309
  32. Davies MJ. 2005. The oxidative environment and protein damage. Biochim. Biophys. Acta 1703: 93-109. https://doi.org/10.1016/j.bbapap.2004.08.007
  33. Shafee T, Gatti-Lafranconi P, Minter R, Hollfelder F. 2015. Handicap-recover evolution leads to a chemically versatile, nucleophile-permissive protease. Chembiochem. 16: 1866-1869. https://doi.org/10.1002/cbic.201500295
  34. Chang Z, Lu M, Ma Y, Kwag DG, Kim SH, Park JM, et al. 2015. Production of disulfide bond-rich peptides by fusion expression using small transmembrane proteins of Escherichia coli. Amino Acids 47: 579-587. https://doi.org/10.1007/s00726-014-1892-y
  35. Nam H, Hwang BJ, Choi DY, Shin S, Choi M. 2020. Tobacco etch virus (TEV) protease with multiple mutations to improve solubility and reduce self-cleavage exhibits enhanced enzymatic activity. FEBS Open Bio 10: 619-626. https://doi.org/10.1002/2211-5463.12828