Abstract
Gelatin is a product obtained through partial hydrolysis and thermal denaturation of collagen, belonging to natural biopeptides. It possesses irreplaceable biological functions in the field of biomedical science and tissue engineering, and has been widely applied. The amino acid sequence of recombinant human gelatin was constructed using a newly designed hexamer composed of six protein monomer sequences in series, with the minimum repeating unit being the characteristic Gly-X-Y sequence found in type III human collagen α1 chain. The nucleotide sequence was subsequently integrated into the genome of Pichia pastoris, leading to successful expression through fermentation. As a result, a recombinant human collagen with a purity exceeding 95% and an expression level of 0.95mg/mL was achieved. Molecular weight determination and amino acid analysis confirmed that the obtained recombinant gelatin had an identical amino acid composition to the one of theoretically designed. Furthermore, scanning electron microscopy revealed that the water-based gel formed by recombinant gelatin exhibited a porous structure. After culturing cells continuously within these gel microspheres for two days followed by fluorescence staining and observation using confocal laser scanning microscopy, it was observed that cells clustered together within the gel matrix, exhibiting three-dimensional growth characteristics while maintaining good viability. This research presents promising prospects for developing recombinant human gelatin as a