Difference between revisions of "Part:BBa K5150006:Design"

 
 
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===Design Notes===
 
===Design Notes===
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The nucleotide sequence encoding the recognition peptide was generated through reverse translation taking into account as a criterion the codon optimization that was useful for bacteria, both for E. coli and for ''Bacillus subtillis''.  
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The nucleotide sequence encoding the recognition peptide was generated through reverse translation taking into account as a criterion the codon optimization that was useful for bacteria, both for ''E. coli'' and for ''Bacillus subtillis''.  
  
  
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===References===
 
===References===
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[1] Raran-Kurussi, S., Cherry, S., Zhang, D., & Waugh, D. S. (2017). Removal of affinity tags with TEV protease. Heterologous Gene Expression in E. coli: Methods and Protocols, 221-230.
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[2] Cesaratto, F., Burrone, O. R., & Petris, G. (2016). Tobacco Etch Virus protease: A shortcut across biotechnologies. Journal of biotechnology, 231, 239-249.
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[3] Boulware, K. T., Jabaiah, A., & Daugherty, P. S. (2010). Evolutionary optimization of peptide substrates for proteases that exhibit rapid hydrolysis kinetics. Biotechnology and bioengineering, 106(3), 339-346.
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[4] Kostallas, G., Löfdahl, P. Å., & Samuelson, P. (2011). Substrate profiling of tobacco etch virus protease using a novel fluorescence-assisted whole-cell assay. PLoS One, 6(1), e16136.

Latest revision as of 09:29, 2 October 2024


TEV protease recognition sequence for tag removal.


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The nucleotide sequence encoding the recognition peptide was generated through reverse translation taking into account as a criterion the codon optimization that was useful for bacteria, both for E. coli and for Bacillus subtillis.


Source

The specific recognition motif for TEV, known as the TEV cleavage site, consists of just 7 amino acids: EXXYXQ-S/G (where X represents any amino acid). Proteolysis occurs between the Gln and Ser or Gly residues. Many studies have demonstrated that within the cleavage site not all residues are equally tolerated. Despite many possible combinations of the cleavage site have been tested, the optimum recognition site is ENLYFQ-S/G, which corresponds to the cleavage site between protein fragments NIb and CP on the natural TEV polyprotein [3]. Despite the sequence of the peptide: ENLYFQS, is identical to the protease's natural substrate peptide, the most commonly use is ENLYFQG [4]. Since a large amount of characterization information has been reported for this sequence, we decided to use it.

References

[1] Raran-Kurussi, S., Cherry, S., Zhang, D., & Waugh, D. S. (2017). Removal of affinity tags with TEV protease. Heterologous Gene Expression in E. coli: Methods and Protocols, 221-230.

[2] Cesaratto, F., Burrone, O. R., & Petris, G. (2016). Tobacco Etch Virus protease: A shortcut across biotechnologies. Journal of biotechnology, 231, 239-249.

[3] Boulware, K. T., Jabaiah, A., & Daugherty, P. S. (2010). Evolutionary optimization of peptide substrates for proteases that exhibit rapid hydrolysis kinetics. Biotechnology and bioengineering, 106(3), 339-346.

[4] Kostallas, G., Löfdahl, P. Å., & Samuelson, P. (2011). Substrate profiling of tobacco etch virus protease using a novel fluorescence-assisted whole-cell assay. PLoS One, 6(1), e16136.