Sengenics is a functional proteomics company that leverages its patented KREXTM technology for production of full-length, correctly folded and functional proteins. KREXTM utilises the Biotin Carboxyl Carrier Protein (BCCP) folding marker which is cloned in-frame with the gene encoding the protein of interest. BCCP acts not only as a protein folding marker but also as a protein solubility enhancer.
BCCP is fused to either the N- or C-terminal of a protein of interest. Full-length proteins are expressed as fusions to the BCCP folding marker which becomes biotinylated in vivo, BUT only when the protein is correctly folded. Conversely, misfolded proteins drive the co-translational misfolding of BCCP to such an extent that it becomes catalytically inactive, and is unable to become biotinylated. Hence, misfolded proteins no longer have a way of attaching to a streptavidin-coated solid support. This is the fundamental principle of the KREXTM technology. Only correctly folded proteins become attached to a solid support.
As biotinylated proteins bound to a streptavidin-coated surface show negligible dissociation, this interaction therefore provides a vastly superior means for tethering proteins to a planar surface and is ideal for applications such as protein arrays, SPR and bead-based assays. The use a compact, folded, biotinylated, 80 residue domain BCCP affords two significant advantages over the AviTag and intein-based tag. First, the BCCP domain is cross-recognised by eukaryotic biotin ligases enabling it to be biotinylated efficiently in yeast, insect, and mammalian cells without the need to co-express the E. coli biotin ligase. Second, the N- and C-termini of BCCP are physically separated from the site of biotinylation by 50Å (Fig. 1), so the BCCP domain can be thought of as a stalk which presents the recombinant proteins away from the solid support’s surface, thus minimising any deleterious effects due to immobilisation.
The success rate for KREXTM mediated expression of even the most complex proteins is in excess of 98%. The technology can be applied in a highly parallelised pipeline resulting in high-throughput, highly consistent production of functionally validated proteins. This ensures that only the correct final 3-dimensional structure is presented on solid supports for truly meaningful biological interactions.
One critical factor that determines the quality of results is the surface chemistry of the solid support the protein is immobilised onto. An ideal surface chemistry should resist non-specific adsorption, provide sufficient stability for the three-dimensional structure of the protein and also provide an environment that allows the protein to retain its native conformation and functionality. As proteins have numerous hydrophobic domains and charged sites, they tend to adsorb non-specifically to most solid surfaces which often results in the disruption of their 3-D structure and eventually, complete loss of activity. This is circumventable using KREXTM. Proteins produced using KREXTM technology can be immobilised onto a wide variety of streptavidin-coated PEG-derivatised solid supports such that they retain folded structure and function in an aqueous environment, and behave in miniaturised, highly multiplexed quantitative assays as if they are in free solution.
Addition of BCCP permits the monitoring of fusion protein folding by measuring the extent of in vivo biotinylation. This can be measured by standard blotting procedures, using SDS-PAGE or in situ colony lysis and transfer of samples to a membrane, followed by detection of biotinylated proteins using a streptavidin conjugate such as streptavidin-horseradish peroxidase.
Additionally, the fact that the BCCP domain is biotinylated in vivo is particularly useful when attempting to multiplex protein purification for fabrication of protein arrays since the proteins can be simultaneously purified from cellular lysates and immobilised in a single step via the high affinity and specificity exhibited by a streptavidin surface.