Expression Studies of the Eukaryotic RHS-repeat Containing Protein Teneurin and its Potential Binding Partner Furin

Abstract

ABC toxins are large bacterial protein complexes involved in the virulence of several invertebrate pathogens. Advances in the structural understanding of ABC toxin complexes, at an atomic levels, have provided unprecedented knowledge regarding the structure and function of the rearrangement-hotspot (RHS)-repeats encoded within the B-C heterodimer of the toxin complex. The RHS-repeats have been found to form an encapsulation device for the cytotoxic component of the C protein which is present at the very C-terminus of the ABC toxin complex. Thus, the RHS-repeats serve to sequester the toxic load, protecting surrounding environments and the toxin producing microorganism from its effects, prior to delivery to host cells. RHS-repeats are widely conserved and found in several species of bacteria as well as the eukaryotic family of proteins, the teneurins. The teneurins are large transmembrane proteins with several established functions both within and outside the central nervous system, where the proteins have been suggested as key regulators of synaptic development and synaptic partner matching. The domain architecture of the teneurin proteins has been found to be remarkably similar to the domain structure of the B-C heterodimer of the ABC toxin complexes. The teneurin proteins possess a bioactive teneurin C-terminal associated peptide (TCAP), reminiscent of the cytotoxic component of the C protein of the ABC toxin complex, which is preceded by a series of functional domains, including the RHS-repeats. This raises the possibility that the RHS-repeats of the teneurins may also form a structure similar to the B-C heterodimer that envelopes the TCAP. This structure could function as a delivery vehicle for the transfer and release of the TCAP into the cytosol of cells, introducing a novel mechanism by which the teneurin proteins may function. The objective of this research was to express and purify a recombinant human teneurin orthologue, human teneurin-1, to gain a structural understanding of the protein. It was expected that determining these details would provide information that would allow the molecular mechanisms by which the teneurin proteins function to be better understood. The expression of recombinant teneurin-1 was investigated using both bacterial and eukaryotic expression systems and proved very challenging. While some progress was made, the difficulties experienced in expressing recombinant protein limited advancement towards the ultimate goal. This research also worked towards the expression and purification of recombinant human furin, a processer and binding partner for the teneurins, to enable functional studies aimed at identifying the interactions existent between teneurins and furin. The protocols required for the expression and purification of recombinant human furin were established using a eukaryotic expression system, which will be used for future interaction studies with recombinant teneurin.