Team:Wageningen UR/Results/affinitybody

Affinity Molecule library

The goal of this project was making the library of affinity molecules to be used in the phage display. The modularity and specificity of the Mantis diagnostic system comes from the use of affinity molecules. These molecules are created by selecting them for their specificity via phage display from a random naive library. The appreaoch taken in this project proved to be a reliable way to create such a library.



Introduction

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 1). 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).

Construct

The vector used to make the library with is pComb3XSS, acquired from AddGene. The pComb3XSS vector has an origin of replication for both e coli and filamentous phage M13. By using the SacI and SpeI restriction sites any protein of interest can be expressed as a fusion to the g3p protein. This protein is incorporated in the M13 helper phages upon infection of bacteria carrying this plasmid.

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.