(715c) Development of Functional Proteinaceous Hydrogels for Biotechnology Applications

Hydrogels consisting of three-dimensional cross-linked polymer networks have emerged as interesting scaffolds for a host of applications including regulated drug delivery, tissue engineering, and biosensors.  Self-assembly of these supramolecular networks is driven by the incorporation of a cross-linking domain into a monomeric building block.  We have used protein engineering to create hydrogels by genetically appending cross-linking domains to globular proteins, which results in unique proteinacous biomaterials that incorporate biological activity [1].  In our previous work, we have investigated α-helical leucine zipper domains to facilitate cross-linking between proteins.  By appending these helices to the N and C termini of globular proteins, we have created hydrogels with benchmark proteins such as GFP [2], and more interestingly, with catalytic enzymes such as organophosphate hydrolase (OPH) [3] and alcohol dehydrogenase (AdhD) [4].  More recently, we have created a hydrogel out of three self-assembling dehygrogenase enzymes which results in a hydrogel that contains a synthetic metabolic pathway that is capable of oxidizing methanol to carbon dioxide [5].  We have now been expanding this approach as we have been extensively characterized a calcium responsive repeats-in-toxin (RTX) domain and evaluated its potential as an alternative cross-linker.  In the absence of calcium, the RTX domain is largely unstructured.  In calcium rich environments, it will reversibly fold into a beta helix secondary structure [6].  By rationally designing one face of the folded beta helix to contain leucine residues, we have created an environmentally-responsive cross-linking domain capable of self-assembly only in the presence of calcium [7].  The folded RTX domain contains a second face amenable to mutation, which we have also designed to contain leucine residues.  We are currently characterizing this “double-face” leucine-rich RTX domain and evaluating is mechanical and biophysical properties using circular dichroism, michrorheology, and multi-angle light scattering.   The RTX domains represent a truly “smart” cross-linking strategy in that the addition of a specific effector molecule (calcium) enables the reversible formation of the hydrogel network. 

1.            Banta, S., I.R. Wheeldon, and M.A. Blenner, Protein engineering in the development of functional hydrogels. Annu Rev Biomed Eng, 2010. 12: p. 176-186.

2.            Wheeldon, I.R., S.C. Barton, and S. Banta, Bioactive proteinaceous hydrogels from designed bifunctional building blocks. Biomacromolecules, 2007. 8(10): p. 2990-4.

3.            Lu, H.D., I.R. Wheeldon, and S. Banta, Catalytic biomaterials: engineering organophosphate hydrolase to form self-assembling enzymatic hydrogels. Protein Eng Des Sel, 2010. 23(7): p. 559-566.

4.            Wheeldon, I.R., E. Campbell, and S. Banta, A chimeric fusion protein engineered with disparate functionalities-enzymatic activity and self-assembly. J Mol Biol, 2009. 392(1): p. 129-42.

5.            Kim, Y.H., et al., Complete oxidation of methanol in biobattery devices using a hydrogel created from three modified dehydrogenases. Angewandte Chemie, 2013. 52(5): p. 1437-40.

6.            Blenner, M.A., et al., Calcium-Induced Folding of a Beta Roll Motif Requires C-Terminal Entropic Stabilization. J Mol Biol, 2010. 400(2): p. 244-256.

7.            Dooley, K., et al., Engineering of an environmentally responsive beta roll peptide for use as a calcium-dependent cross-linking domain for peptide hydrogel formation. Biomacromolecules, 2012. 13(6): p. 1758-64.