K. Durrant
Utah State University, Utah, United States
Keywords: Recombinant hagfish protein, neural tissue engineering, biomaterials, neurite outgrowth, cell adhesion, GRGGL motif, neuroprotection, high-yield protein expression, neuroblastoma cells, reactive oxygen species, mitochondrial membrane potential, intermedi
We present a novel biomaterial based on engineered recombinant hagfish intermediate filament alpha (rHIF-α) protein, designed to support neuronal growth and regeneration. By incorporating the spider silk-derived GRGGL repeat, a neural cell adhesion molecule (NCAM) binding motif, we developed two constructs: a full-length protein (full-GR) and a central rod domain variant (CRD-GR). Both proteins were expressed at high yields (7.76 g/L and 12.63 g/L, respectively) and purified to over 90% purity, significantly outperforming previous silk-inspired protein yields. N2a neuroblastoma cells cultured on these engineered protein films demonstrated high viability and enhanced neurite outgrowth compared to native-like sequence rHIF-α and PLL controls, with cells on full-GR exhibiting the most extensive neurite development. Investigation of neuroprotective properties revealed intriguing patterns in reactive oxygen species levels and mitochondrial membrane potential, suggesting complex interactions between the engineered protein films and cellular redox balance. Our findings highlight the potential of these engineered rHIF-α proteins as high-yield, bioactive substrates for neural tissue engineering, offering promising avenues for treating neural injuries and neurodegenerative diseases.