470417 Engineering New Allosterically - Regulated Protein Interactions for Applications in Biotechnology
We evaluated the potential of this peptide as a cross-linker for hydrogel formation. We showed that calcium dependent hydrogel formation can be achieved by designing the two faces of the folded beta helix to contain leucine residues. When there is no calcium, β-roll domains remain unstructured, delocalizing the leucine rich patches. After calcium binding, the β-roll folds and the leucine rich faces are exposed creating a hydrophobic driving force for self-assembly1. We created various constructs where we used the leucine rich β-roll mutant as a hydrogel building block. In addition, we showed that this peptide can serve as a new scaffold for biomolecular recognition. We randomized the amino acid residues on one of the beta sheet forming faces and selected mutants, which demonstrated an affinity for various targets, including lysozyme. Isothermal titration calorimetry (ITC) was utilized to quantitatively determine the thermodynamic parameters of interactions in solution between the mutants and their target. In the case of the lysozyme library, we achieved sub-micromolar / mid-nanomolar (0.52μM - 63nM) dissociation constants. We also performed affinity chromatography experiments and showed that the best mutant is capable of capturing its target, in the presence of calcium2. The reversibility of the calcium binding allows for the engineered molecular recognition to be controllable. The captured target is easily eluted upon removal of the calcium ions. This will be of great interest in the bio-separation area as well as other areas of biotechnology, where binding events can be easily and reversibly controlled.
- K. Dooley, B. Bulutoglu and S. Banta, Doubling the cross-linking interface of a rationally-designed beta roll peptide for calcium-dependent proteinaceous hydrogel formation. Biomacromolecules, 2014, 15(10): 3617-3624.
- B. Bulutoglu, K. Dooley, M. Blenner and S. Banta, Catch & Release: Fishing for target proteins with an evolved β-roll peptide exhibiting allosteric regulation, submitted.
See more of this Group/Topical: Food, Pharmaceutical & Bioengineering Division