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.
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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. The paper also shows that using this technique it is possible to estimate the pI values for a wide range of proteins measuring at only two pH values, suggesting that this technique is rapid and accurate on small volume samples.įigure 1: shows a diagram of the microfluidic chip used to determine the pI of the target proteins without a spatial pH gradient and instead using a temporal gradient.Cell-adhesive factors mediate adhesion of cells to substrates via peptide motifs such as the Arg–Gly–Asp (RGD) sequence. The technique requires low voltages and low sample consumption. The paper shows that this method is successful in determining the pI using this new technique without the requirement of generating and maintaining pH gradients which is often challenging for other techniques. To demonstrate the effectiveness of this method the pI of 7 different proteins of known pI were tested β-lactoglobulin, ribonuclease A, ovalbumin, human transferrin, ubiquitin and myoglobin. The approach exploits temporal rather than spatial pH gradients. using a microfluidic system built in house design a new technique to determine a protein’s isoelectric point (pI) based on microfluidic free-flow electrophoresis (μFFE).
#DETERMINATION OF ISOELECTRIC POINT FREE#
The ability to conduct measurements in free solution thus provides the basis for the rapid determination of isoelectric points of proteins under a wide variety of solution conditions and in small volumes.
To demonstrate the general approachability of this platform, they have measured the isoelectric points of representative set of seven proteins, bovine serum albumin, β-lactoglobulin, ribonuclease A, ovalbumin, human transferrin, ubiquitin and myoglobin in microlitre sample volumes. In particular, in this approach, the pH of the electrolyte solution is modulated in time rather than in space, as in the case for conventional determinations of the isoelectric point. Here, Łapińska et al., introduce a gradient-free approach, exploiting a microfluidic platform which allows us to perform rapid pH change on chip and probe the electrophoretic mobility of species in a controlled field. The majority of conventional methods for the determination of the isoelectric point of a molecule rely on the use of spatial gradients in pH, although significant practical challenges are associated with such techniques, notably the difficulty in generating a stable and well controlled pH gradient. The isoelectric point (pI) of a protein is a key characteristic that influences its overall electrostatic behavior.
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