Coding

Part:BBa_K1159100

Designed by: Dong-Jiunn Jeffery TRUONG   Group: iGEM13_TU-Munich   (2013-09-14)

Autolysis resistant TEV Protease S219V mutant in RFC[25]

TEV Protease from Tabacco Etch Virus (TEV) is a highly specific protease that cleaves the sequence "Glu-Asn-Leu-Tyr-Phe-Gln-(Gly/Ser) (ENLYFQ(G/S))" very specifically before the last Gly/Ser. The wildtype version of the protease has a short half-life due to self-inactivation through autolysis. This mutant S219V is autolysis resistant and its enzymatic activity is not affected by the mutation. This part is flanked by RFC[25] pre- and suffix for further protein fusions.

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
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 316
    Illegal SapI.rc site found at 664

Protein data table for BioBrick BBa_ automatically created by the BioBrick-AutoAnnotator version 1.0
Nucleotide sequence in RFC 25, so ATGGCCGGC and ACCGGT were added (in italics) to the 5' and 3' ends: (underlined part encodes the protein)
 ATGGCCGGCGGAGAAAGC ... CTCATGAATACCGGT
 ORF from nucleotide position -8 to 714 (excluding stop-codon)
Amino acid sequence: (RFC 25 scars in shown in bold, other sequence features underlined; both given below)
1 
101 
201 		MAGGESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNKHLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQK
LKFREPQREERICLVTTNFQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGSPLVSTRDGFIVGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQ
WVSGWRLNADSVLWGGHKVFMVKPEEPFQPVKEATQLMNTG*
Sequence features: (with their position in the amino acid sequence, see the list of supported features)
Factor Xa cleavage site: 80 to 83
Amino acid composition:
Ala (A)5 (2.1%)
Arg (R)10 (4.1%)
Asn (N)15 (6.2%)
Asp (D)10 (4.1%)
Cys (C)4 (1.7%)
Gln (Q)13 (5.4%)
Glu (E)10 (4.1%)
Gly (G)19 (7.9%)
His (H)8 (3.3%)
Ile (I)14 (5.8%)
Leu (L)17 (7.1%)
Lys (K)14 (5.8%)
Met (M)8 (3.3%)
Phe (F)17 (7.1%)
Pro (P)14 (5.8%)
Ser (S)20 (8.3%)
Thr (T)22 (9.1%)
Trp (W)5 (2.1%)
Tyr (Y)3 (1.2%)
Val (V)13 (5.4%)
Amino acid counting
Total number:241
Positively charged (Arg+Lys):24 (10.0%)
Negatively charged (Asp+Glu):20 (8.3%)
Aromatic (Phe+His+Try+Tyr):33 (13.7%)
Biochemical parameters
Atomic composition:C1216H1874N334O355S12
Molecular mass [Da]:27237.0
Theoretical pI:8.90
Extinction coefficient at 280 nm [M-1 cm-1]:31970 / 32220 (all Cys red/ox)
Plot for hydrophobicity, charge, predicted secondary structure, solvent accessability, transmembrane helices and disulfid bridges 
Codon usage
Organism:E. coliB. subtilisS. cerevisiaeA. thalianaP. patensMammals
Codon quality (CAI):good (0.70)good (0.71)good (0.68)good (0.76)excellent (0.81)good (0.68)
Alignments (obtained from PredictProtein.org)
SwissProt:P04517 (99% identity on 237 AAs), Q85197 (57% identity on 236 AAs)
TrEML:Q000U0 (98% identity on 237 AAs), G9IZP9 (96% identity on 237 AAs)
PDB:1lvm (99% identity on 219 AAs)
Predictions (obtained from PredictProtein.org)
Subcellular Localization (reliability in brackets)
Archaea:secreted (100%)
Bacteria:cytosol (41%)
Eukarya:cytosol (19%)
Gene Ontology (reliability in brackets)
Molecular Function Ontology: -
Biological Process Ontology:GO:0006508 (47%), GO:0019079 (47%)
 
Predicted features:
Disulfid bridges: -
Transmembrane helices: -
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Categories
Parameters
None