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<p> | <p> | ||
| − | + | The modularity and specificity of the Mantis diagnostic system comes from the use of affinity molecules. Affinity molecules are | |
| − | + | antibody mimetics based on staphylococcal protein A (SPA) (Nord et al., 1997).The small, 6kDa, affinity proteins are based on the | |
| − | + | Z domain of the cell-wall anchored bacterial protein A. The native function of protein A is immunoglobin binding and contributes | |
| − | + | to evading the immune system (Nord et al., 1995). By making changes to 13 amino acids on 2 helices essential for specificity, | |
| − | + | affibodies for a wide variety of targets can be developed (Figure X). Since its discovery affibodies have been developed for targets | |
| − | + | such as insulin, fibrinogen, transferrin, tumor necrosis factor-a, IL-8, gp120, CD28, human serum albumin, IgA, IgE and HER2 | |
| − | + | (Löfblom et al., 2010). Potential uses for these affibodies are imaging, purification, detection and many therapeutic applications | |
| − | + | (Löfblom et al., 2010).</p> | |
</div></section> | </div></section> | ||
Revision as of 12:50, 23 October 2017
Affinity Molecule library
The modularity and specificity of the Mantis diagnostic system comes from the use of affinity molecules. Affinity molecules are antibody mimetics based on staphylococcal protein A (SPA) (Nord et al., 1997).The small, 6kDa, affinity proteins are based on the Z domain of the cell-wall anchored bacterial protein A. The native function of protein A is immunoglobin binding and contributes to evading the immune system (Nord et al., 1995). By making changes to 13 amino acids on 2 helices essential for specificity, affibodies for a wide variety of targets can be developed (Figure X). Since its discovery affibodies have been developed for targets such as insulin, fibrinogen, transferrin, tumor necrosis factor-a, IL-8, gp120, CD28, human serum albumin, IgA, IgE and HER2 (Löfblom et al., 2010). Potential uses for these affibodies are imaging, purification, detection and many therapeutic applications (Löfblom et al., 2010).
Introduction
The proteins that are selected to be used for the phage display are surface proteins from Trypanosomes. The selection was made based on the titre in the blood and the reactivity to IgG in earlier findings. The surface of Trypanosomes is mostly covered in the Lille Trypanosoma Antigen Type Variant Surface Glycoprotein (LiTat VSG). This makes it unsuitable to be used as the protein to which our test is based upon. The Invariant Surface Glycoprotein (ISG) is also a surface antigen of Trypanosomes. There are several forms, of different sizes. Of those, ISG64 (64 kDa) and ISG65 (65 kDa) are the most reactive towards antibodies for both forms of HAT, 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 [2]. This will simplify the expression and purification process, but keeping the immunogenicity. Although there are a 100 VSG for every ISG, its genetic stability is adventurous [3].
Construct
The three ISG antigens suitable as a biomarker for HAT are PCR amplified from genomic Trypanosoma brucei DNA. Genomic DNA from Trypanosoma brucei gambiense is 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 are PCR amplified using gene-specific primers extended with a 5’ KpnI and SacI restriction sites. This is followed by cloning into the Multiple Cloning Site (MSC) of the E. coli expression vector pET52b via restriction digestion with KpnI and SacI. This resulted in a recombinant gene (rISG) with a 5’ Strep-tag II and a 3’ 10x HIS-tag. The construct is present under a IPTG-inducible promotor, see figure 1.
This construct is transformed to E. coli DH5α. The constructs were checked with colony PCR and sequencing. The sequence of the construct was compared to the sequence of the original template, as well as the reference sequence from online databases. Whereas the sequence of rISG64 and rISG65 could be validated, the one of rISG75 could not. Too many unexplainable mismatches were found to continue with protein expression. One explanation could be that ISG75 is part of a gene family, and a family member has been amplified. Because the correctness of the sequence could not be verificated, this construct was not further used.
After this validation step, the two remaining plasmids were transformed to E. coli Rosetta for protein expression. This strain contains the pRARE plasmid, having extra tRNA genes compromising for the 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, see figure 2.
As seen, protein expression could be induced, where the protein is present in the soluble fraction as expected.
Protein purification
Next, 200 ml cultures were grown, following by induction with 0.5 mM IPTG. Protein purification was conducted by affinity purification in gravity columns using strep-tactin, making use of the StrepII-tag. Purity was checked on SDS gel, and protein concentration in the eluted fractions was measured using a protein quantitation assay. All protocols can be found on link to protocol section and lab journal.
The extracellular domain of the Invariant Surface Glycoprotein 64 and 65, fused to both a StrepII-tag and 10x HIS-tag has succesfully been purified using strep-tactin gravity column, see figure 3.
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. As seen from the high amounts of protein in the flowthrough, the column has reached its saturation point.
These tagged proteins, bound to the strep-tactin beads, are used for phage display selection.
Moreover, two biobricks were created of these constructs: BBa_K2387060 and BBa_K2387061. For this, the recombinant ISG gene, including the two tags, was cloned into the linearized pSB1C3 vector using biobrick assembly.
References
- Biéler, Sylvain, et al. "Evaluation of Antigens for Development of a Serological Test for Human African Trypanosomiasis." PloS one 11.12 (2016): e0168074.
- 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.
- Overath, P., et al. "Invariant surface proteins in bloodstream forms of Trypanosoma brucei." Parasitology Today 10.2 (1994): 53-58.
