Optimization of Extraction and Isolation of Proteins from Eggshells
Authors: Richter Ryan , Lee Eun Joo , Grant Jennifer E. 
Published: June 2021

The eggshells of the common chicken (Gallus gallus domesticus) are a large waste product of the food industry; thus, developing methods for harvesting bioactive proteins from eggshells is of great economic interest. Eggshells are composed of a calcium carbonate mineral structure along with up to a 3 to 5 % organic fraction by mass (Nys et al., 2004, General Paleontology, 3: 549–562). While the mineral fraction has already found widespread use in agriculture as a fertilizer and more recently as a catalyst for biodiesel production, the protein fraction, has yet to be fully utilized. Before eggshells can be utilized, however, the egg shell inner and outer membranes must be removed. For large scale membrane separations, several industrial processes involving the use of pressurized gases, multiphase floatation tanks or liquid cavitation are available. However, these methods are not practical for small scale exploratory preparations.For small‐scale protein extractions from egg shells, various incubation solutions for membrane removal were assessed using individual eggshells. Raw eggshells were incubated in solutions of 1 M HCl, 1 % SDS, 50 mM EDTA and reverse osmosis‐treated water. The ease of which membranes could be separated from eggshells manually was assessed. Once a membrane removal procedure was established, the optimal method was used for further experimentation. Membrane‐less eggshells were then rinsed with a 1 % SDS solution. The remaining shell was then dissolved in a minimum volume of 10 % acetic acid solution. After dialysis of the resulting solution, micro‐chromatography techniques were employed to isolate specific fractions of protein. Bradford assays were performed between each step to assess total protein content, while SDS‐PAGE using 12% and tricine gels were performed to evaluate the sizes of proteins recovered.Removing the entire membrane at once is important as it greatly reduces the potential of leaving behind small portions of membrane protein, which would contaminate the extract. Several published protocols for separating inner and outer egg shell membranes have called for chemical mixtures of 1 M HCl, weak EDTA solutions or SDS. However, prolonged exposure causes the membrane itself to weaken and tear, causing small fragments of membrane to remain attached to the shell. After comparing the results of the varying incubation solutions, simply using a large quantity of water proved to be the most effective. Using aqueous solutions hydrates the membranes, causing them to separate from the shell more easily and reduces tearing. Using water has the additional benefit of reducing chemical waste. Following this step an SDS wash was used to lightly remove any remaining contamination.Once isolated from the shell, the total protein fraction was enriched for phosphoproteins using micro‐chromatographic techniques involving Ni2+‐affinity resin and/or hydroxyapatite chromatography. Additionally, ammonium sulfate fractionation was used to divide the total protein fraction into discrete protein fractions by ammonium sulfate cuts of 40%, 60% and 80%.Support or Funding InformationThis research was supported by a University of Wisconsin‐Stout Student Research Grant to RR.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.