Biotechnological Control of Hydrogel Properties via Recombinant Protein Molecular Weight Engineering

Hydrogels based on natural polymers are widely used in 3D cell culture and tissue engineering due to their biocompatibility and tunability. In this work, recombinant collagen-derived proteins of defined molecular weights were designed and tested as precursors for methacrylated, photocrosslinkable hydrogels. Proteins of 25.6 kDa, 58 kDa, and 89.2 kDa were recombinantly expressed in Komagataella phaffii, methacrylated, and photocrosslinked to form well-defined hydrogels. A Design of Experiments (DoE) strategy was employed to quantify the effects of degree of functionalization (DoF) and precursor molecular weight on hydrogel stiffness, deformability, and swelling. For the first time, it was reported that both the DoF and molecular weight of recombinant proteins used for hydrogel fabrication significantly influence hydrogel properties. The molecular weight effects were most pronounced at lower chain lengths. Predictive models generated from the DoE revealed non-linear and interactive contributions of both parameters, while mixed-material formulations suggested non-additive behavior beyond the fitted design space. Additionally, biocompatibility for all materials was shown by live-dead staining of cells seeded onto the crosslinked materials. The results demonstrate that recombinant protein chain length can be used as a powerful design parameter to modulate hydrogel mechanics. Such materials not only enable xeno-free cultivation but also provide a biotechnological route toward rationally engineered biomaterials for diverse applications.