Team:Wageningen UR/Results/Trypanosoma

Parasitic Antigens

The aim of this project is to express antigens from the African Sleeping Sickness parasite, also called Human African Trypanosomiasis (HAT), in Escherichia coli (E. coli) and to subsequently purify these antigens. This part functions as a proof-of-concept showing that, besides viral infections, Mantis can detect parasitic infections as well. The project resulted in the successful expression of double-tagged, extracellular domains of the recombinant surface glycoproteins rISG64 and rISG65. Consequently, these soluble proteins have been purified using the Strep-tag II. Finally, rISG64 is used for the selection of Affinity Molecules via phage display.


Introduction

Surface proteins from Trypanosoma brucei gambiense, the causative agent of HAT, were selected to be used for the phage display. The selection was made based on the titer in the blood and the reactivity to IgG in earlier findings [1]. The surface of Trypanosomes is mostly covered in the Lille Trypanosoma Antigen Type Variant Surface Glycoprotein (LiTat VSG). However, since the sequence of this protein is highly variable and changes every few generations [2] it is unsuitable to be used as the protein to which our test is based upon. Another surface antigen is the Invariant Surface Glycoprotein (ISG) which comes in different forms and sizes. Of those, ISG64 (64 kDa) and ISG65 (65 kDa) are the most reactive towards anti-HAT antibodies, followed by the 75 kDa ISG75 [1]. By removing the signal peptide on the N-terminal and the transmembrane domain on the C-terminal, a soluble protein is created [3]. This will simplify the expression and purification process but will keep the immunogenicity. Even though ISG is a 100 times less abundant on the parasite’s membrane, it is more genetically stable compared to VSG [2].


The three ISG antigens suitable as a biomarker for HAT were PCR amplified from genomic Trypanosoma brucei gambiense DNA. Genomic DNA from T. brucei was made available by the WHO Collaborating Center for Research and Training on Human African Trypanosomiasis Diagnostics in Antwerp. The extracellular domains of ISG64, ISG65 and ISG75 were PCR amplified using gene-specific primers flanked with KpnI and SacI restriction sites. This is followed by cloning into the Multiple Cloning Site (MCS) of the E. coli expression vector pET52b(+) via restriction digestion with KpnI and SacI. This resulted in a recombinant gene (rISG) containing a 5’ Strep-tag II and a 3’ 10x HIS-tag under a lac promoter, see Figure A.

Figure A: Map of the recombinant ISG genes after cloning into the pET52b expression vector.

This construct is transformed into E. coli DH5α. The constructs were checked using colony PCR and verified via sequencing. The sequences of the constructs were compared to the sequence of the original template. The sequence of rISG64 and rISG65 could be validated, but the one of rISG75 could not due to too many mismatches. Therefore, rISG75 was used in further purification steps.

After sequence validation, the two remaining plasmids were transformed to E. coli Rosetta™(DE3) for protein expression. This strain contains the pRARE plasmid, having extra tRNA genes for the expression of any rare codons present in the parasitic genome.


Protein expression

The induction of protein expression of the pET52b-ISG constructs was tested, as well as the solubility of the recombinant proteins. For this, a small-scale 3 ml culture was used. Cell extracts before and after IPTG induction were run on SDS gel, as well as from the soluble and insoluble fractions, see Figure 1. The protocol can be found on the protocol page. As seen in Figure 1, protein expression could be induced, where the protein is present in the soluble fraction as expected.

Figure 1: SDS Gel of cell lysate before and after IPTG induction, as well as the soluble and insoluble fraction hereof. The bands at the expected sizes for rISG64 (42.0 kDa) and rISG65 (45.4 kDa) are indicated with the red box.


Protein purification

Next, the cultures are upscaled to 200 ml. This was followed by induction with 0.5 mM IPTG. Protein purification was conducted by affinity purification in gravity columns using strep-tactin sepharose beads, which bind to the Strep-tag II. Purity was checked on SDS gel and protein concentration in the eluted fractions was measured using the Roti-Nanoquant protein quantitation assay. More can be read in the notebook.

The extracellular domain of the Invariant Surface Glycoprotein 64 and 65, fused to both a Strep-tag II and 10x HIS-tag has successfully been purified using strep-tactin gravity column, see Figure 2.

Figure 2: SDS gel of the protein fractions eluted from the strep-tactin column. Both the flow through after loading the cell lysis onto the column, a few washing steps and the elution fractions can be seen for rISG64 (top) and rISG65 (bottom). Expected sizes: 42.0 kDa (rISG64) and 45.4 kDa (rISG65).

The final 50 μl elution fraction (Elute 4) contains 283 μg/ml protein for rISG64, whereas the elution for rISG65 just contains 63 μg/ml protein. Therefore, rISG64 was chosen to be used for phage display selection. To this end, the protein bound to the strep-tactin beads is used to select affinity bodies with high affinity for rISG64.


BioBrick production

Besides, the two recombinant ISG genes, including the two tags, were cloned individually into the linearized pSB1C3 vectors using the BioBrick assembly. This resulted in two new BioBricks, BBa_K2387060 and BBa_K2387061.

References

  1. Biéler, Sylvain, et al. "Evaluation of Antigens for Development of a Serological Test for Human African Trypanosomiasis." PloS one 11.12 (2016): e0168074.
  2. Overath, P., et al. "Invariant surface proteins in bloodstream forms of Trypanosoma brucei." Parasitology Today 10.2 (1994): 53-58.
  3. 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.