Improvement in the Orthogonal Protein Degradation in Escherichia coli by Truncated mf-ssrA Tag

  • Lu Lv
  • Yang Wu
  • Guozhen Zhao
  • Hao QiEmail author
Research Article


SsrA peptide tag from Mycoplasma florum has been developed as a versatile biotechnology tool to control orthogonal degradation of tagged proteins in Escherichia coli. Here, using the systematic deletion mutants of mf-ssrA tag, we demonstrated that the residues in two separate regions have different functions in mf-Lon-mediated specific orthogonal target protein degradation in E. coli. The deletion of multiple residues, up to six amino acids, did not fatally abolish its specific degradation activity, instead of being able to improve the stability of the tagged protein in the presence of endogenous proteases before mf-Lon expression in E. coli. Except for previously identified essential residues, the region adjacent to the C-terminal of the mf-ssrA tag was involved in mf-Lon and endogenous protease-mediated degradation. Moreover, the deletion of specific residues made the mf-ssrA tag more effective and compact. The mf-ssrA tag can be implemented in synthetic biology and bioengineering for development of synthetic circuits.


mf-ssrA Protein degradation Escherichia coli L region R region 



This work was supported by the National Natural Science Foundation of China (No. 21476167 and No. 21778039).


  1. 1.
    Ambro L, Pevala V, Bauer J et al (2012) The influence of ATP-dependent proteases on a variety of nucleoid-associated processes. J Struct Biol 179(2):181–192CrossRefGoogle Scholar
  2. 2.
    Botos I, Melnikov EE, Cherry S et al (2004) Crystal structure of the AAA + alpha domain of E. coli Lon protease at 1.9 A resolution. J Struct Biol 146(1–2):113–122CrossRefGoogle Scholar
  3. 3.
    Farrell CM, Grossman AD, Sauer RT (2005) Cytoplasmic degradation of ssrA-tagged proteins. Mol Microbiol 57(6):1750–1761CrossRefGoogle Scholar
  4. 4.
    Hari SB, Sauer RT (2016) The AAA + FtsH protease degrades an ssrA-tagged model protein in the inner membrane of Escherichia coli. Biochemistry 55(40):5649–5652CrossRefGoogle Scholar
  5. 5.
    Moore SD, Sauer RT (2007) The tmRNA system for translational surveillance and ribosome rescue. Annu Rev Biochem 76:101–124CrossRefGoogle Scholar
  6. 6.
    Lies M, Maurizi MR (2008) Turnover of endogenous ssrA-tagged proteins mediated by ATP-dependent proteases in Escherichia coli. J Biol Chem 283(34):22918–22929CrossRefGoogle Scholar
  7. 7.
    Gur E, Sauer RT (2008) Evolution of the ssrA degradation tag in Mycoplasma: specificity switch to a different protease. Proc Natl Acad Sci USA 105(42):16113–16118CrossRefGoogle Scholar
  8. 8.
    Baneyx F, Mujacic M (2004) Recombinant protein folding and misfolding in Escherichia coli. Nat Biotechnol 22(11):1399–1408CrossRefGoogle Scholar
  9. 9.
    Gottesman S, Roche E, Zhou Y et al (1998) The ClpXP and ClpAP proteases degrade proteins with carboxy-terminal peptide tails added by the ssrA-tagging system. Genes Dev 12(9):1338–1347CrossRefGoogle Scholar
  10. 10.
    Bittner LM, Arends J, Narberhaus F (2016) Mini review: ATP-dependent proteases in bacteria. Biopolymers 105(8):505–517CrossRefGoogle Scholar
  11. 11.
    Dougan DA, Weber-Ban E, Bukau B (2003) Targeted delivery of an ssrA-tagged substrate by the adaptor protein sspB to its cognate AAA + Protein ClpX. Mol Cell 12(2):373–380CrossRefGoogle Scholar
  12. 12.
    Karzai AW, Susskind MM, Sauer RT (2014) SmpB, a unique RNA-binding protein essential for the peptide-tagging activity of ssrA (tmRNA). EMBO J 18(13):3793–3799CrossRefGoogle Scholar
  13. 13.
    Chien P, Grant RA, Sauer RT et al (2007) Structure and substrate specificity of an sspB ortholog: design implications for AAA + adaptors. Structure 15(10):1296–1305CrossRefGoogle Scholar
  14. 14.
    Cameron DE, Collins JJ (2014) Tunable protein degradation in bacteria. Nat Biotechnol 32(12):1276–1281CrossRefGoogle Scholar
  15. 15.
    Chan CTY, Lee JW, Cameron DE et al (2016) ‘Deadman’ and ‘Passcode’ microbial kill switches for bacterial containment. Nat Chem Biol 12(2):82–86CrossRefGoogle Scholar
  16. 16.
    Maier JAH, Möhrle R, Jeltsch A (2017) Design of synthetic epigenetic circuits featuring memory effects and reversible switching based on DNA methylation. Nat Commun 8:15336CrossRefGoogle Scholar
  17. 17.
    Zhang C, Tsoi R, You L (2016) Addressing biological uncertainties in engineering gene circuits. Integr Biol 8(4):456–464CrossRefGoogle Scholar
  18. 18.
    Wang HH, Church GM (2011) Multiplexed genome engineering and genotyping methods applications for synthetic biology and metabolic engineering. Methods Enzymol 498:409–426CrossRefGoogle Scholar
  19. 19.
    Flynn JM, Levchenko I, Seidel M et al (2001) Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis. Proc Natl Acad Sci USA 98(19):10584–10589CrossRefGoogle Scholar
  20. 20.
    Chien P, Perchuk BS, Laub MT et al (2007) Direct and adaptor-mediated substrate recognition by an essential AAA + protease. Proc Natl Acad Sci USA 104(16):6590–6595CrossRefGoogle Scholar
  21. 21.
    Novoa PG, Williams KP (2004) The tmRNA website: reductive evolution of tmRNA in plastids and other endosymbionts. Nucleic Acids Res 32(32):104–108CrossRefGoogle Scholar
  22. 22.
    Lessner FH, Venters BJ, Keiler KC (2007) Proteolytic adaptor for transfer-messenger RNA-tagged proteins from α-proteobacteria. J Bacteriol 189(1):272–275CrossRefGoogle Scholar
  23. 23.
    Wiegert T, Schumann W (2001) SsrA-mediated tagging in Bacillus subtilis. J Bacteriol 183(13):3885–3889CrossRefGoogle Scholar

Copyright information

© Tianjin University and Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.School of Chemical Engineering and TechnologyTianjin UniversityTianjinChina

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