A literature search turns up no more than a few reports on RGD-containing plant proteins, such as the renin-like protein in the cardoon, Cynara cardunculus 4. One approach toward identifying potential cell-adhesive proteins in nature would be to look for proteins that contain the Arg–Gly–Asp (RGD) motif, which was the first cell-adhesive peptide sequence to be identified and remains the most widely applied 1, 3. Ideally, these cell culture support materials are non-animal-derived and edible, such that they do not need to be dissociated from the propagated cells and form part of the food product, thus saving time and cost. Some of the cell types employed in these nascent industries, such as muscle cells, are anchorage-dependent, requiring adhesion to a culture support material in order to survive and proliferate. In recent years, sustainability and animal welfare concerns have given rise to an endeavour to replace animal products with those obtained from cells grown in culture e.g., cell-cultivated meat, milk, and liver 2. They have the advantage of being biocompatible, in addition to possessing native ligands that can be recognized and bound by a wide range of mammalian cell types. The common biomaterials used for mammalian cell adhesion are animal-derived extracellular proteins such as collagen and fibronectin. In tissue engineering, for example, such materials are important to mediate adhesion of various cell types to scaffolds for tissue or organ regeneration 1. A method has been developed to identify candidate species and produce cell-adhesive matrices, applicable to the cell-cultivated food and healthcare industries.Ĭell-adhesive materials, specifically materials that can support the attachment, spreading, proliferation, and differentiation of cells are widely used in the biomedical and pharmaceutical industries. Therefore, a sustainable source of cell-adhesive proteins is widely available in the fungi kingdom. A snapshot of the RGD-containing proteins in the fungal extracts was obtained by combining SDS-PAGE and mass spectrometry of the peptide fragments obtained by enzymatic cleavage. We demonstrated a cell traction stress on the protein particles (from Flammulina velutipes) that was comparable to cells on fibronectin. These protein particles were incorporated in 3D fiber matrices encapsulating mouse myoblast cells, showing a positive effect on cell alignment. We observed the formation of protein particles in crude extracts isolated from basidiomycete fungi, which could be correlated to their stability towards particle aggregation at different temperatures. A plot of fungi species vs RGD percentage revealed that 98% of the species exhibited an RGD percentage > = 1%. Screening of a protein database for fungal and plant proteins uncovered that ~5.5% of the unique reported proteins contain RGD sequences. In this paper, we show how data mining can be a powerful approach toward identifying fungal-derived cell-adhesive proteins and present a method to isolate and utilize these proteins as extracellular matrices (ECM) to support cell adhesion and culture in 3D. With the onset of sustainability issues, there is a pressing need to find alternatives to animal-derived cell-adhesive factors, especially for cell-cultivated food applications. Cell-adhesive factors mediate adhesion of cells to substrates via peptide motifs such as the Arg–Gly–Asp (RGD) sequence.
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