Difference between revisions of "Part:BBa K2387060"

 
 
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<partinfo>BBa_K2387060 short</partinfo>
 
<partinfo>BBa_K2387060 short</partinfo>
  
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This part contains the extracellular domain (domain 24-365) of the invariant surface glycoprotein 64 (ISG64) of Trypanosoma brucei gambiense. This parasite causes African Sleeping Sickness (Human African Trypanosomiasis). ISG64 is a surface protein and thus antigen of this organism. It is double tagged, having a Strep-tag II and 10xHis tag for the convenience of purification. Moreover, because of two distinct cleavage sites, the tags can be cleaved of if necessary.  
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This part contains the extracellular domain of the invariant surface glycoprotein 64 (ISG64) of <i>Trypanosoma brucei gambiense</i>: [https://parts.igem.org/Part:BBa_K2387058 BBa_K2387058]. 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 [https://parts.igem.org/Part:BBa_K2387056 BBa_K2387056] and [https://parts.igem.org/Part:BBa_K2387057 BBa_K2387057]).
  
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By only taking the extracellular domain, protein expression and purification are simplified since a soluble protein is formed. 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.  
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===Usage and Biology===
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The <i>Trypanosoma brucei gambiense</i> parasite causes African Sleeping Sickness (Human African Trypanosomiasis, HAT). ISG64 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. ISG64 is evenly distributed over the cell surface of trypanosomes, of around 10^4-10^5 copies per cell [1].
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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 24-365 ([http://www.uniprot.org/uniprot/C9ZQ37 Uniprot C9ZQ37]).  
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Note that this DNA originates from parasitic genomic DNA, and codon optimization has not been performed. Hence, it is advised to use an <i>E. coli</i> 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.   
 
This protein can be used for, for example, a proof-of-principle for the binding to or detection of African Sleeping Sickness.   
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===Usage and Biology===
 
  
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K2387060 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2387060 SequenceAndFeatures</partinfo>
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===Protein expression and purification===
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This part was used under an IPTG-inducible promoter (T7 promoter/lac operator) in a high copy plasmid in <i>E. coli Rosetta</i> strain.
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By experiments, it was confirmed that induction of protein expression under this promotor was successful, and indeed a soluble protein is created.
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Purification was performed using a gravity column with strep-tactin beads, having affinity for the Strep-tag II.
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[[File:Expression_rISG64.jpg|500px|center|thumb|<p align="justify">'''Figure 1: Expression and purification of rISG64 in <i>E. coli Rosetta</i>. Expected size: 42.0 kDa.'''</p>]]
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===References===
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#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.
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# 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.
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Latest revision as of 13:19, 1 November 2017


Double tagged ISG64 of T.b. gambiense

This part contains the extracellular domain of the invariant surface glycoprotein 64 (ISG64) of Trypanosoma brucei gambiense: BBa_K2387058. 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). ISG64 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. ISG64 is evenly distributed over the cell surface of trypanosomes, of around 10^4-10^5 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 24-365 ([http://www.uniprot.org/uniprot/C9ZQ37 Uniprot C9ZQ37]). 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
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 811

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 rISG64 in E. coli Rosetta. Expected size: 42.0 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.