Recombinant Protein Scaffolds for Tissue Engineering

The introduction of chemically unique groups into proteins by means of non-natural amino acids has numerous applications in protein engineering and functional studies. One method to achieve this involves the utilization of a non-natural amino acid by the cell's native translational apparatus. Here we demonstrate that a methionine surrogate, azidohomoalanine, is activated by the methionyl-tRNA synthetase of Escherichia coli and replaces methionine in proteins expressed in methionine-depleted bacterial cultures. We further show that proteins containing azidohomoalanine can be selectively modified in the presence of other cellular proteins by means of Staudinger ligation with triarylphosphine reagents. Incorporation of azide-functionalized amino acids into proteins in vivo provides opportunities for protein modification under native conditions and selective labeling of proteins in the intracellular environment.

Recombinant DNA methods were used to create artificial proteins that undergo reversible gelation in response to changes in pH or temperature. The proteins consist of terminal leucine zipper domains flanking a central, flexible, water-soluble polyelectrolyte segment. Formation of coiled-coil aggregates of the terminal domains in near-neutral aqueous solutions triggers formation of a three-dimensional polymer network, with the polyelectrolyte segment retaining solvent and preventing precipitation of the chain. Dissociation of the coiled-coil aggregates through elevation of pH or temperature causes dissolution of the gel and a return to the viscous behavior that is characteristic of polymer solutions. The mild conditions under which gel formation can be controlled (near-neutral pH and near-ambient temperature) suggest that these materials have potential in bioengineering applications requiring encapsulation or controlled release of molecular and cellular species.

Spider silks are protein-based "biopolymer" filaments or threads secreted by specialized epithelial cells as concentrated soluble precursors of highly repetitive primary sequences. Spider dragline silk is a flexible, lightweight fiber of extraordinary strength and toughness comparable to that of synthetic high-performance fibers. We sought to "biomimic" the process of spider silk production by expressing in mammalian cells the dragline silk genes (ADF-3/MaSpII and MaSpI) of two spider species. We produced soluble recombinant (rc)-dragline silk proteins with molecular masses of 60 to 140 kilodaltons. We demonstrated the wet spinning of silk monofilaments spun from a concentrated aqueous solution of soluble rc-spider silk protein (ADF-3; 60 kilodaltons) under modest shear and coagulation conditions. The spun fibers were water insoluble with a fine diameter (10 to 40 micrometers) and exhibited toughness and modulus values comparable to those of native dragline silks but with lower tenacity. Dope solutions with rc-silk protein concentrations >20% and postspinning draw were necessary to achieve improved mechanical properties of the spun fibers. Fiber properties correlated with finer fiber diameter and increased birefringence.