Expression, characterization, and application of human-like recombinant gelatin

• Published in: Bioresources and bioprocessing Vol. 11; no. 1; pp. 69 - 13
• Main Authors: Song, Xiaoping, Chu, Tao, Shi, Wanru, He, Jingyan
• Format: Journal Article
• Language: English
• Published: Singapore Springer Nature Singapore 17.07.2024; Springer Nature B.V; SpringerOpen
• Subjects: Amino acid composition; Amino acid sequence; Amino acids; Biochemical Engineering; Biomedical engineering; Biomedical materials; Characterization; Chemistry; Chemistry and Materials Science; Collagen; Collagen (type III); Confocal microscopy; Environmental Engineering/Biotechnology; Fermentation; Gelatin; Gene expression; Industrial and Production Engineering; Microscopy; Microspheres; Molecular structure; Molecular weight; Monomer protein sequence; Nucleotide sequence; Nucleotides; Pichia pastoris; Porous media; Protein engineering; Recombinant human gelatin; Scanning electron microscopy; Scanning microscopy; Thermal denaturation; Thermodynamic properties; Three- dimensional culture; Tissue engineering; Characterization; Monomer protein sequence; Biomedical materials; Recombinant human gelatin; Three- dimensional culture; Pichia pastoris
• ISSN: 2197-4365; 2197-4365
• DOI: 10.1186/s40643-024-00785-1

Gelatin is a product obtained through partial hydrolysis and thermal denaturation of collagen, belonging to natural biopeptides. With irreplaceable biological functions in the field of biomedical science and tissue engineering, it has been widely applied. The amino acid sequence of recombinant human-like gelatin was constructed through 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 inserted into the genome of Pichia pastoris to enable soluble secretion expression of recombinant gelatin. At the shake flask fermentation level, the yield of recombinant gelatin is up to 0.057 g/L, and its purity can rise up to 95% through affinity purification. It was confirmed in the molecular weight determination and amino acid analysis that the amino acid composition of the obtained recombinant gelatin is identical to that of the theoretically designed. Furthermore, scanning electron microscopy revealed that the freeze-dried recombinant gelatin hydrogel exhibited a porous structure. After culturing cells continuously within these gelatin microspheres for two days followed by fluorescence staining and observation through confocal laser scanning microscopy, it was observed that cells clustered together within the gelatin matrix, exhibiting three-dimensional growth characteristics while maintaining good viability. This research presents promising prospects for developing recombinant gelatin as a biomedical material.