Production of a Recombinant Non-Hydroxylated Gelatin Mimetic in Pichia pastoris for Biomedical Applications

Abstract

Proteins derived from the natural extracellular matrix like collagen or gelatin are common in clinical research, where they are prized for their biocompatibility and bioactivity. Cells are able to adhere, grow and remodel scaffolds based on these materials. Usually, collagen and gelatin are sourced from animal material, risking pathogenic transmission and inconsistent batch-to-batch product quality. A recombinant production in yeast circumvents these disadvantages by ensuring production with a reproducible quality in animal-component-free media. A gelatin mimetic protein, based on the alpha chain of human collagen I, was cloned in Pichia pastoris under the control of the methanol-inducible alcohol oxidase (AOX1) promoter. A producing clone was selected and cultivated at the 30 L scale. The protein was secreted into the cultivation medium and the final yield was 3.4 g·L−1. Purification of the target was performed directly from the cell-free medium by size exclusion chromatography. The gelatin mimetic protein was tested in cell culture for biocompatibility and for promoting cell adhesion. It supported cell growth and its performance was indistinguishable from animal-derived gelatin. The gelatin-mimetic protein represents a swift strategy to produce recombinant and human-based extracellular matrix proteins for various biomedical applications.

Expression, characterization, and application potentiality evaluation of recombinant human-like collagen in Pichia pastoris

“…3 a) below 200 nm (198 nm in this study) indicated that the RHLC had triple helical characteristics (Salvatore et al 2020 ). As the RHLC consisted of a triple-helix structure and did not have a terminal domain similar to extracted collagen, no significant peak appeared at 220 nm that displayed a beta-sheet (Gellermann et al 2019 ; Gibney et al 2021 ; Zhang et al 2019 ). The FTIR results (Fig.…”

Cosmetic Potential of a Recombinant 50 kDa Protein

“…Many previous studies have tried to produce recombinant collagen molecules in different systems including bacteria, yeast and plant [25,[28][29][30][31]37,[43][44][45][46]. These recombinant proteins include full-length or fragments of collagen I, II, III and XXI at different hydroxylation levels (unhydroxylated or hydroxylated).…”

“…In the last two decades, more and more effort has been put into recombinant collagen production as part of the rapid development of genetic engineering technology. Recombinant collagen molecules of different sizes have been expressed in all major expression platforms including mammalian cells, insect cells, yeast, bacteria and plant cells [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37]. In general, high quality full-length collagen proteins have been produced in eukaryotic hosts but with very low productivity.…”

Three Decades of Research on Recombinant Collagens: Reinventing the Wheel or Developing New Biomedical Products?

“…Furthermore, researchers investigated plants, including tobacco, barley, and corn, as potential collagen-producing factories. Experiments with animals demonstrated the feasibility of producing collagens in the mammary glands of transgenic mice and eggs of transgenic chickens [ 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 ].…”