Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: IC 2010 Team
Date created: 2010-10-22 11:00:00
Date modified: 2015-05-08 01:11:56
TEV protease S219P autocatalysis resistant variant
| Types | DnaRegion |
| Roles | Coding CDS |
| Sequences | BBa_K316012_sequence (Version 1) |
Description
TEV protease S219P autocatalysis resistant variantIntroduction :
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 http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-45PMGK3-9P&_user=217827&_coverDate=02%2F01%2F1994&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000011279&_version=1&_urlVersion=0&_userid=217827&md5=e075aad3a349720ad9484095d01a65be&searchtype=a] 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 specificity [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-45PMGK3-9P&_user=217827&_coverDate=02%2F01%2F1994&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000011279&_version=1&_urlVersion=0&_userid=217827&md5=e075aad3a349720ad9484095d01a65be&searchtype=a] 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 219 [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WXR-45R86R6-5X&_user=7635175&_coverDate=06%2F20%2F1995&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000011279&_version=1&_urlVersion=0&_userid=7635175&md5=0a1521e783c0bd4126b145f3f6d766d4&searchtype=a]
. 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)
Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency
Table I. Kinetic parameters for wild-type and mutant TEV proteases with the peptide substrate TENLYFQSGTRR-NH2.
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
S219P* - virtually imperivious to autocatalysis
S219V* - retains same activity as wild type
Full article can be seen here [http://peds.oxfordjournals.org/content/14/12/993.long]
Notes
The part was produced by nucleotide synthesis by mwg ??? eurofinSource
TEV protease is naturally found in Tobacco Etch Virus genome. This part contains the sequence codon optimized for expression in B.subtilis using mwg ??? eurofins [[www.eurofinsdna.com]] proprietary software.| Sequence Annotation | Location | Component / Role(s) |
| TEV protease | 1,717 | CDS feature/protein |