Team:Wageningen UR/Results/Cpx BiFC

Antigen visualization using BiFC

In order to visualize antigen binding, we use E. coli's Cpx two-component signal transduction pathway. We combine its protein-protein interactions with Bimolecular Fluorescence Complementation and show how we obtain rapid and specific visual response upon activation!

We now know the native function of E. coli's Cpx two-component signal transduction pathway, and how we can use this system to detect and bind antigens (Link to ./Results/Cpx_System.html). However, we need to create to create an output signal to visually show the presence of antigen. To do so we can use the native protein-protein interactions of the Cpx pathway to our advantage.


We use visualization method called Bimolecular Fluorescence Complementation (BiFC). BiFC is based on the association of fragments of a fluorescent reporter protein fused to interacting target proteins [1] (Figure 1). A fluorophore can be split into two non-fluorescent fragments which reassemble into a fluorescent complex upon interaction between the aforementioned target proteins. BiFC can directly be used in living cells, and does not need addition of substrates. Another advantage is that a simple photo spectrometer is enough to measure the signal.

Visualising Cpx interactions

We use several protein interactions of the Cpx pathway to visualize antigen binding. Upon activation of the Cpx pathway, CpxP gets titrated away from CpxA which is activated and autophosphorylates. This phosphogroup is then transferred to CpxR, which can then homodimerize [REF]. By fusing split reporter proteins to these Cpx-proteins, Cpx pathway activation can directly be visualized! We decided to link CpxR dimerization (figure 2A) and CpxA-CpxR interaction (Figure 2B) to eYFP-termini, which are often used in BiFC.

A third possible method uses specific cleavage via TEV protease. TEV is fused to CpxR whereas eYFPn and eYFPc are fused to CpxA. The eYFP-termini are fused to antiparallel leucine zippers. Upon Cpx activation, CpxA and CpxR interact and the eYFP-Zipper fusions are cleaved off of CpxA and they can freely move through the cytoplasm. The leucine zippers have natural affinity for each other and will act as target proteins in BiFC and facilitate eYFP recomplementation (Figure 2C)

Figure 2: The BiFC rationale: Target proteins A and B are fused to split reporter Y (YN and YC). Once A and B interact, YN and YC reassembly and regain fluorescence[REF1].

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.

Figure 1: Map of the recombinant ISG genes after clonation into the pET52b expression vector.

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.

Figure 2: SDS Gel of cell lysate before and after IPTG induction, as well as the soluble and insoluble fraction hereof. The assumed bands for rISG64 and rISG65 are indicated with the red box.

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.

Figure 3: SDS gel of the protein fractions eluted from the strep-tactin column, both the flowthrough after loading the cell lysis onto the column, a few washing steps and the elution fractions.

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

  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. 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.
  3. Overath, P., et al. "Invariant surface proteins in bloodstream forms of Trypanosoma brucei." Parasitology Today 10.2 (1994): 53-58.