tetanus toxoid (TT), and artificial carrier-systems, e.g. Commonly used carrier proteins are keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) and rabbit serum albumin (RSA), but also other proteins, e.g. Usually peptide-carrier conjugates for AB production are formed by coupling an about 20 amino acid-long peptide to residues at the surface of a carrier protein via a chemical reaction 8, 9, 10. For B-cell activation, the conjugated peptide needs to be exposed on the surface of the carrier, and it is thought that a dense packing of the conjugated peptide is advantageous for this, because highly repetitive epitopes on the particle/carrier surface facilitate B-cell receptor oligomerization 1, 2. For the recognition of the protein in its native form, the correct sequence, but also the structure and surface exposure of the selected peptide need to be considered 7.
For the reactivity of the ABs, the selection of the peptide is critical, since the ABs derived from the conjugate can only recognize the peptide as presented (or similar) on the carrier 7. by immunofluorescence imaging or western blotting. Such ABs can be used to identify proteins, which contain the peptides used for AB generation, in complex samples, and allow the specific labelling of proteins of interest in their spatiotemporal distribution, e.g. One application of scaffold proteins is their conjugation with peptides for the generation of antibodies (AB), utilizing the increased immunogenicity of the protein-peptide conjugate (in this role often called carrier proteins) 6. Scaffolds need to form a stable and water-soluble structure that is best insensitive to the attached cargo, and they should further allow the dense packing of the cargo in homovalent and ideally also in heterovalent fashion 1, 2, 3, 4, 5. Two key features are obligatory, in addition. Depending on the actual use, multiple features of the protein can be important e.g., particle size, achievable purity, expression level, robustness of fold/assembly, general stability and immunogenicity (if used for immunizations). In a case study, we finally show that mtDod-peptide fusions allow producing antibodies against human heat shock proteins and the C-terminus of heat shock cognate 70 interacting protein (CHIP).įor being suited as scaffolds, proteins need to meet an array of requirements. Further, mtDod equipped with SYNZIP and Sp圜atcher domains for post-translational recruitment of cargo was prepared of which the mtDod/Sp圜atcher system proved to be particularly useful. with monomeric superfolder green fluorescent protein creating a 437 kDa large dodecamer, were successfully purified, showing mtDod’s ability to function as recruitment hub. Fusions of mtDod with proteins of up to four times the size of mtDod, e.g. We show that mtDod, either directly loaded with cargo or equipped with domains for non-covalent and covalent loading of cargo, can be produced recombinantly in high quantity and quality in Escherichia coli. MtDod is a homododecameric complex of spherical shape, high stability and robust assembly, which allows the attachment of cargo at its surface. Here, we present the dodecin from Mycobacterium tuberculosis ( mtDod) as a new scaffold protein. For example, scaffolds can be used to make weak or non-immunogenic small molecules immunogenic by attaching them to the scaffold, in this role called carrier. In bioengineering, scaffold proteins have been increasingly used to recruit molecules to parts of a cell, or to enhance the efficacy of biosynthetic or signalling pathways.