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Nature-based Peptide and Protein Scaffolds as Enzyme Mimetics
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Protein and peptide scaffolds are of interest for many bioengineering applications. The incorporation of catalytic activity in such structures is of major importance, although it represents a challenge. Several strategies are being used to obtain peptide catalysts and protein/peptide-based hydrogels, but the merging of the two fields is still in infancy. This work aims to study a new peptide scaffold, based on the small domain peptide, which is part of PTEN, a tumor suppressor protein. To fully characterize this peptide scaffold, the peptide was produced by two strategies; (i) small domain fused with GFP (SD-GFP) and; (ii) the small domain without fusion partner (SD). Both strategies were developed by using bacterial cells as hosts and purified by chromatographic-based techniques. The soluble SD-GFP scaffold was obtained with 93% purity, and showed catalytic activity towards pNPP, presenting a rate of 5x10-4 s-1, which is an order of magnitude lower than the kcat of PTEN towards pNPP. The production of SD-GFP also led to the formation of inclusion bodies, which were solubilized and matrix-assisted refolded on-column with 69% purity, leading to a self-supporting hydrogel at 4 ºC. The gel was washed by using three cycles of PBS and distilled water, which allowed to increase hydrogel purity. This corroborates the fact that hydrogel network is being formed by SD-GFP. The SD peptide was produced in the soluble form in bacterial cells and was purified yielding 90% of purity. The presence of paired cysteines in the SD peptide sequence lead to the formation of disulfide bridges forming tetrameric assemblies. These assemblies present an α-helical structure but are not catalytically active. In the future, the peptide should be in its reduced form (in monomer), so that cysteines can act as a nucleophile, catalyzing the dephosphorylation of pNPP.
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catalysis peptide PTEN scaffold self-assembly