Tag

Part:BBa_K3781019:Design

Designed by: Nicolas Bayer   Group: iGEM21_TU_Kaiserslautern   (2021-10-08)


GST + TEV Motif, MocloMania B5


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 628
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 628
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 628
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 628
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

This basic part was de novo synthesized and thus easily codon optimized towards Leishmania tarentolae.
We generated this part by designing it in silico to carry the sequence of interest flanked by two invertedly oriented BbsI recognition sites as well as the desired B3 overhangs. After commercial synthesis, this allowed us to introduce the part into its respective plasmid backbone with a simple MoClo ligation.


Source

The genetic sequence for this tag was derived from a publically available Addgene plasmid named pET-GST, #42049.
The most common version of the GST-tag originally derives its sequence from the gluthatione-S-transferase coding gene in Schistosoma japonicum, a parasitic plathelminthes. This is because the first ever implementation of GST for affinity purification was by Smith et al. who proposed the worm's GST protein as a possible vaccine against schistosomiasis.[1] The TEV protease motif sequence is based on the recognition sequence of the tobacco etch virus TEV protease. The plant virus encodes its whole genome as one cohesive polyprotein and endogenous proteases such as TEV protease help to liberate individual functional protein units. Thus, its recognition sequence can be found throughout the tobacco etch virus genome.[2]


References

  1. ↑ Smith DB, Johnson KS. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31-40. doi: 10.1016/0378-1119(88)90005-4. PMID: 3047011.
  2. ↑ Carrington JC, Dougherty WG (May 1988). "A viral cleavage site cassette: identification of amino acid sequences required for tobacco etch virus polyprotein processing". Proc. Natl. Acad. Sci. U.S.A. 85 (10): 3391–5. Bibcode:1988PNAS...85.3391C. doi:10.1073/pnas.85.10.3391. PMC 280215. PMID 3285343.