Gelatin hydrogels find broad medical application. The current materials, however, are from animal sources, and their molecular structure and thermal properties cannot be controlled. This study describes recombinant gelatin-like polymers with a general design that inherently offers independent tuning of the cross-link density, melting temperature, and biocompatibility of the gel. The polymers contain small blocks with thermoreversible trimerization capacity and defined melting temperature, separated by hydrophilic nontrimerizing blocks defining the distance between the knot-forming domains. As an example, we report the secreted production in yeast at several g/L of two nonhydroxylated approximately 42 kDa triblock copolymers with terminal trimerizing blocks. Because only the end blocks formed cross-links, the molecular architecture of the gels is much more defined than that of traditional gelatins. The novel hydrogels had a approximately 37 degrees C melting temperature, and the dynamic elasticity was independent of the thermal history. The concept allows to produce custom-made precision gels for biomedical applications.

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We studied the influence of molecular size on the formation of transient networks by telechelic protein polymers with $2.3 kDa collagen-like triple helix-forming end-blocks and much longer random coil mid-blocks. We compared triblock copolymers with mid-blocks of $400 and $800 amino acids (37 and 73 kDa, respectively) and two different amino acid sequences, all of which were secreted to high concentration by recombinant yeast cells. At the same molar concentration of protein and crosslinkforming end-blocks, the storage modulus of the longer polymers was higher than that of the shorter polymers. Differences in storage modulus values were also observed for the polymers with mid-blocks of the same amino acid composition but different amino sequence, which correlated to differences in the measured hydrodynamic radius of the mid-blocks. The melting temperature of the triple helices was the same for both larger and smaller polymers; however, the elastic properties of the gels were lost at lower temperature for the smaller polymers than for the larger polymers. Using an analytical model based on classical gel theory and accounting for the well-defined multiplicity of the network, we could ascribe these differences to the lower probability of the longer chains to form intramolecular loops.

Previously, we have shown that gel-forming triblock proteins, consisting of random coil middle blocks and trimer-forming (Pro-Gly-Pro)(9) end blocks, are efficiently produced and secreted by the yeast Pichia pastoris. These end blocks had a melting temperature (T(m)) of ∼41°C (at 1.1 mM of protein). The present work reveals that an increase of T(m) to ∼74°C, obtained by extension of the end blocks to (Pro-Gly-Pro)(16), resulted in a five times lower yield and partial endoproteolytic degradation of the protein. A possible cause could be that the higher thermostability of the longer (Pro-Gly-Pro)(16) trimers leads to a higher incidence of trimers in the cell, and that this disturbs secretion of the protein. Alternatively, the increased length of the proline-rich (Pro-Gly-Pro)(n) domain may negatively influence ribosomal translation, or may result in, for example, hydrophobic aggregation or membrane-active behavior owing to the greater number of closely placed proline residues. To discriminate between these possibilities, we studied the production of molecules with randomized end blocks that are unable to form triple helices. The codon- and amino acid composition of the genes and proteins, respectively, remained unchanged. As these nontrimerizing molecules were secreted intact and at high yield, we conclude that the impaired secretion and partial degradation of the triblock with (Pro-Gly-Pro)(16) end blocks was triggered by the occurrence of intracellular triple helices. This degradation was overcome by using a yapsin 1 protease disruptant, and the intact secreted polymer was capable of forming self-supporting gels of high thermal stability.