A novel epitope-presenting thermostable scaffold for the development of highly specific insulin-like growth factor-1/2 antibodies.

High sequence and structural homology between mature human insulin-like growth factors IGF-1 and IGF-2 makes serological discrimination by immunodiagnostic IGF tests a challenging task. There is an urgent need for highly specific IGF-1 and IGF-2 antibodies, yet only a short sequence element, i.e. the IGF loop, provides enough difference in sequence to discriminate between the two molecules. We sought to address this unmet demand by investigating novel chimeric immunogens as carriers for recombinant peptide motif grafting. We found Thermus thermophilus sensitive to lysis D (SlyD) and Thermococcus gammatolerans SlyD FK-506-binding protein (FKBP) domains suitable for presentation of the predefined epitopes, namely the IGF-1 and IGF-2 loops. Chimeric SlyD-IGF proteins allowed for the development of exceptionally specific IGF-1 and IGF-2 monoclonal antibodies. The selected antibodies bound with high affinity to the distinct IGF epitopes displayed on the protein scaffolds, as well as on the mature human IGF isoforms. The respective SlyD scaffolds display favorable engineering properties in that they are small, monomeric, and cysteine-free and can be produced in high yields in a prokaryotic host, such as Escherichia coli In conclusion, FKBP domains from thermostable SlyD proteins are highly suitable as a generic scaffold platform for epitope grafting.

[Fe4S4]- and [Fe3S4]-cluster formation in synthetic peptides.

[Fe4S4]- and [Fe3S4]-clusters are ubiquitous iron-sulfur motifs in biological systems. The [Fe3S4] composition is, however, of much lower natural abundance than the more typical [Fe4S4]-clusters. In the present study formation of [Fe3S4]-clusters has been examined using chemically synthesized model peptides consisting of 33 amino acids (maquettes). Maquettes are effective synthetic analogs for metal-ion binding sites, allowing for a facile modification of the primary coordination sphere of iron-sulfur clusters. Maquettes have been designed following the [FeS]-cluster-binding motif of dimethyl sulfoxide reductase subunit B (DmsB) from Escherichia coli that carries a [Fe4S4]-cluster, but incorporates a [Fe3S4]-cluster instead upon mutation of one of the coordinating cysteines. The time-dependent formation of iron-sulfur clusters and the effects of exchanging selected amino acids in the model peptides, known to regulate the [Fe3S4] to [Fe4S4] ratio in the DmsB protein, were monitored by UV/Vis- and EPR-spectroscopy. Exchange of cysteines within the conserved CxxCxxC motif has a much stronger effect on cluster formation and stoichiometry than the exchange of a coordinating external cysteine. Amino acid exchange in the binding motif shows a dependence of the cluster stoichiometry on the amino acid side chain. Formation of [Fe3S4]-clusters in maquettes is less favorable compared to native proteins. The [Fe3S4] moiety appears to be a rather transient species towards the more stable (final) incorporation of a [Fe4S4]-cluster. Results are best described by an assembly mechanism that considers a successive coordination of the iron atoms by the peptide, rather than incorporation of an already pre-formed mercaptoethanol-coordinated [Fe4S4]-cluster.