Difference between revisions of "Part:BBa K2387061"
 
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Purification was performed using a gravity column with strep-tactin beads, having affinity for the Strep-tag II.  
 
Purification was performed using a gravity column with strep-tactin beads, having affinity for the Strep-tag II.  
  
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[[File:Expression_rISG65.jpg|500px|center|thumb|<p align="justify">'''Figure 1: Expression and purification of rISG65 in <i>E. coli Rosetta</i>'''</p>]]
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[[File:Expression_rISG65.jpg|500px|center|thumb|<p align="justify">'''Figure 1: Expression and purification of rISG65 in <i>E. coli Rosetta</i>. Expected size: 45.4 kDa.'''</p>]]
  
  

Latest revision as of 13:19, 1 November 2017


Double tagged ISG65 of T.b. gambiense

This part contains the extracellular domain of the invariant surface glycoprotein 65 (ISG65) of Trypanosoma brucei gambiense: BBa_K2387059. It is double tagged, having an N-terminal Strep-tag II and C-terminal 10xHis tag for the convenience of purification. Moreover, because of two distinct cleavage sites, the tags can be cleaved of if necessary (see BBa_K2387056 and BBa_K2387057).

Usage and Biology

The Trypanosoma brucei gambiense parasite causes African Sleeping Sickness (Human African Trypanosomiasis, HAT). ISG65 is a surface protein and thus antigen of this organism. The main feature of the ISG proteins is the conserved sequence, it does not undergo antigenic variation. This is making it a suitable target for diagnostics and therapeutics. ISG65 is evenly distributed over the cell surface of trypanosomes, of around 7-5x 10^4 molecule copies per cell [1].

By only taking the extracellular domain, protein expression and purification are simplified since a soluble protein is formed [2]. For this, the signal peptide and transmembrane domain are removed, leaving residues 21-385. ([http://www.uniprot.org/uniprot/Q26712 Uniprot Q26712]). Note that this DNA originates from parasitic genomic DNA, and codon optimization has not been performed. Hence, it is advised to use an E. coli strain optimized for this.

This protein can be used for, for example, a proof-of-principle for the binding to or detection of African Sleeping Sickness.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 823
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Protein expression and purification

This part was used under an IPTG-inducible promoter (T7 promoter/lac operator) in a high copy plasmid in E. coli Rosetta strain.

By experiments, it was confirmed that induction of protein expression under this promotor was successful, and indeed a soluble protein is created.

Purification was performed using a gravity column with strep-tactin beads, having affinity for the Strep-tag II.

Figure 1: Expression and purification of rISG65 in E. coli Rosetta. Expected size: 45.4 kDa.


References

  1. Jackson, David G., Henry J. Windle, and H. Paul Voorheis. "The identification, purification, and characterization of two invariant surface glycoproteins located beneath the surface coat barrier of bloodstream forms of Trypanosoma brucei." Journal of Biological Chemistry 268.11 (1993): 8085-8095.
  2. Sullivan, Lauren, et al. "Proteomic selection of immunodiagnostic antigens for human African trypanosomiasis and generation of a prototype lateral flow immunodiagnostic device." PLoS neglected tropical diseases 7.2 (2013): e2087.


5 Summary: