Difference between revisions of "Part:BBa K316012"
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'''Auto-inactivation:'''
 
'''Auto-inactivation:'''
  
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Wild type TEV protease also cleaves itself at Met 218 and Ser 219<cite>2</cite>. This leads to auto-inactivation of the TEV protease and progressive loss of activity of the protein. The rate of inactivation is proportional to the concentration of protease<cite>2</cite>
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Wild type TEV protease also cleaves itself at Met 218 and Ser 219<cite>2</cite>. This leads to auto-inactivation of the TEV protease and progressive loss of activity of the protein. The rate of inactivation is proportional to the concentration of protease. More stable Mutants have been produced by single amino acid substitutions S219V (AGC(serine) to GTG(valine) and S219P (AGC(serine) to CCG(proline)<cite>2</cite>.
  
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More stable Mutants have been produced by single amino acid substitutions S219V (AGC(serine) to GTG(valine) and S219P (AGC(serine) to CCG(proline)
 
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Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency
 
  
 
=== '''Table I.''' ===
 
=== '''Table I.''' ===

Revision as of 16:26, 24 October 2010

TEV protease S219P autocatalysis resistant variant


TEV protease S219P autocatalysis resistant variant. This part had been reversed for the 3' strand in order to reduce any read-through that may be caused by upstream elements. Expression of TEV protease is under control of enhanced hyperspank promoter BBa_K316000.


Introduction :

This is the nuclear inclusion protease, endogenous to Tobacco Etch Virus and is used in the late lifecycle to cleave polyprotein precursors. The recognition sequence is ENLYFQG/S 1 between QG or QSDue to it’s stringent sequence specificity, TEV is commonly used to cleave genetically engineered proteins.


Uses:

TEV proteinase is used to cleave fusion proteins. It is useful due to its high degree of specificity1 and potential to be used in vivo or in vitro applications.


Auto-inactivation:

Wild type TEV protease also cleaves itself at Met 218 and Ser 2192. This leads to auto-inactivation of the TEV protease and progressive loss of activity of the protein. The rate of inactivation is proportional to the concentration of protease. More stable Mutants have been produced by single amino acid substitutions S219V (AGC(serine) to GTG(valine) and S219P (AGC(serine) to CCG(proline)2.


Table I.

Kinetic parameters for wild-type and mutant TEV proteases with the peptide substrate TENLYFQSGTRR-NH2. From original paper by Kapust et.al. 20013

Enzyme Km (mM) kcat (s-1) kcat /Km (mM-1 s-1)
Wild type 0.061 ± 0.010 0.16 ± 0.01 2.62 ± 0.46
S219V 0.041± 0.010 0.19 ± 0.01 4.63 ± 1.16
S219P 0.066 ± 0.008 0.09 ±0.01 1.36 ± 0.22

S219V* - retains same activity as wild type

S219P* - virtually imperivious to autocatalysis



Sequence and Features



Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 32
  • 1000
    COMPATIBLE WITH RFC[1000]



References

<biblio>

  1. 1 pmid=8179197
  2. 2 pmid=7793070
  3. 3 pmid=2047602

</biblio>


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