Salt precipitation can be a very powerful tool to purify proteins by precipitation. Ammonium sulfate is usually the salt of choice since it is cheap, very soluble in water, and is able to become much more hydrated interacts with more water molecules than almost any other ionic solvent. In practice, ammonium sulfate is either added directly as a solid or added as a usually saturated solution to precipitate desired proteins. This happens because the dissolved salt competes with the proteins for scarce water molecules, increasing the surface tension of water and therefore causing the protein to fold tighter.
The reduction in protein surface area means less protein-water interactions which allows for more hydrophobic interactions between protein molecules, causing aggregation and subsequently precipitation. Proteins in solution can also be fractionated out since they will precipitate out as a function of salt concentration.
In this way it is possible to purify specific proteins by adding a specific amount of ammonium sulfate to precipitate out non-desirable proteins, recovering the supernatant, and then adding a bit more ammonium sulfate to precipitate out the desired protein and then save that pellet of precipitated protein. Since salt precipitation only affects the solubility of proteins and does not denature them, the recovered fraction can be stored in the salt solution for prolonged periods of time without having to worry about bacterial contamination since the high salt content inhibits any microbial growth or protease activity.
Because ammonium sulfate precipitation only reduces the solubility of proteins and does not denature them proteins can be concentrated by removing the remaining ammonium sulfate solution then the protein pellet can be resolubilized in standard buffers or a lower concentration of ammonium sulfate.
In addition, most subunit vaccines produced in these systems are heat sensitive and require parenteral delivery, which restricts the use of recombinant subunit vaccines in many developing countries where health systems are not well equipped Rigano and Walmsley, For use in plant molecular biofarming, the tobacco plant has several advantages over other plants, such as highly efficient transformation and regeneration, relatively short period for biomass production, and easy homogenization processing for protein purification due to soft leaf tissue Jamal et al.
The tumor-associated antigen GA, a glycoprotein that is highly expressed on the cell-surface of colorectal carcinomas, has previously been fused with the human immunoglobulin Fc fragment carrying the ER retention sequence, to produce the recombinant antigen-antibody complex GAFcK in a plant expression system Lu et al.
Moreover, expression of the recombinant protein GAFcK has been successful in a plant system Lim et al. Optimization of the protein purification process is essential for the successful production of the tagged recombinant therapeutic protein in the total soluble protein TSP recovery step during the downstream process Geyer et al. To purify the tagged recombinant proteins, the TSPs must be extracted, separated, and eventually isolated, from the plant biomass debris Desai et al.
In this study, a recombinant colorectal cancer vaccine candidate fusion protein GAFcK expressed in transgenic Nicotiana tabacum plants was purified by protein-G affinity chromatography. Thus, we optimized ammonium sulfate TSP precipitation conditions to increase the recovery rate of GAFcK in transgenic plant leaves. Seedlings of transgenic tobacco N. Schematic diagram of downstream processing of recombinant protein GAFcK from plant leaf biomass. After removal of the chloroplasts, the pH of the solution was brought up to 7.
The supernatant of the extracted sample was then filtered through a 0. After washing the column, the protein was eluted with elution buffer 1 M glycin-HCl, pH 2. The proteins electrophoresed through the gel were transferred to a nitrocellulose membrane Millipore Corp. Protein bands were visualized by exposing the membrane to an X-ray film Fuji, Tokyo, Japan using a chemiluminescence substrate Pierce. No band was observed in the non-transgenic plant NT.
TSP precipitation in the plant leaf extraction solutions was visualized on a Coomassie-stained gel. We confirmed and optimized the second ammonium sulfate concentration for a downstream procedure as above. Fractions 2 and 3 showed strong protein band signals Figure 4. Black arrowhead indicates a 65 kDa GAFc protein band.
Plants used as expression systems for the production of recombinant therapeutic proteins are considered to have several advantages over other expression systems. These include low production cost of biomass containing recombinant proteins. However, the downstream process, including extraction and purification of the recombinant proteins from plant biomass, often is more complicated and expensive than other fermentation systems. Plant tissues contain a wide range of proteins with different properties, and these portions are subjected to extraction and purification during downstream processing.
The non-optimized purification steps involved in recovering recombinant proteins expressed in plants increase the production cost of plant-derived proteins. However, existing purification procedures for these recombinant proteins have not been fully optimized Lu et al. Contact Us. International Distributors. Description Ammonium sulfate precipitation is one of the most commonly used methods for protein purification from a solution.
Protocol Allow serum or ascitic fluid to thaw, determine total volume, and centrifuge at g for 30 minutes. Transfer sample to a beaker containing a stir bar and place on a magnetic stirrer.
Volume of ammonium sulfate needed is equal to the volume of sample.
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