The separation of important molecules from complex solutions is often accomplished using proteins or peptides that have been engineered to bind the target with high affinity and selectivity. One challenge in this approach is the recovery of the target, and reuse of the binding peptide. The PI has been working with a unique peptide (called the beta roll) that is unstructured in the absence of calcium, and folds into a flattened corkscrew shape in the presence of calcium. The PI has previously engineered one face of the corkscrew for self-assembly, and has preliminary data showing the peptide can be engineered to bind to a model target protein (lysozyme). The goal of this NSF project is to develop a high throughput method to engineer new beta roll mutants that can bind to different protein targets that are important in biotechnology. The incorporation of these new engineered peptides into a bioseparations platform would be very beneficial, as it would allow for target proteins to be bound in the presence of calcium and then released upon calcium removal. This process could improve performance and reduce the costs associated with critical protein molecules, especially therapeutic proteins.
One of the key challenges in affinity-based separations is the elution of the target molecule from the affinity binding reagent. The PI proposes to expand the directed evolution approach to substantially increase the affinity of the beta roll peptides to desired targets and expand the number of targets for molecular recognition.
A method for selection from a randomized library has been developed, but higher affinity binders will require a directed evolution approach where genetic diversity is incorporated into the library. New beta roll peptides with high affinity for GFP and two common protein expression and purification tags will be researched: the maltose binding proteins (MBP) and the glutathione S transferase protein (GST). By immobilizing these evolved, high-affinity beta roll peptides on a suitable support, the PI may be able to demonstrate the use of these peptides to affinity purify MBP- and GST-tagged proteins, and use calcium chelation to elute the purified proteins. The resin can be regenerated via calcium addition and the performance of this system can be compared to traditional methods for purification with these fusion tags (amylose resin and GSH resin).
The Intellectual Merit of this proposal results from the use of a unique peptide with an intrinsic triggered conformational change as a starting scaffold for the engineering of biomolecular recognition. The calcium-induced conformational change of the beta roll peptides is a powerful molecular switch that can be exploited to reversibly disrupt engineered biomolecular interactions. Using this peptide as a starting scaffold, we will be able to generate a collection of peptides that can bind target proteins in a calcium-dependent fashion, and these new peptides will be valuable biomolecular recognition elements for affinity bioseparations.
The Broader Impact of this proposal arises from the use of protein engineering to develop a new binding motif for use in applications such as biosensors and bioseparations. The use of intrinsically disordered scaffolds for biomolecular recognition has not yet been reported in the literature, and this proposal will demonstrate that these systems can be engineered to be high affinity binders, which will be boon to those working in areas such as biosensors, smart drug delivery, bionanotechnology, and bioseparations. Funding will also be used for the mentoring of students and to continue existing outreach activities in the local community.
