Special Considerations When Extracting Plant Proteins

There is simply no other way around it. You need to break down the walls (the cell wall, that is) to extract the good stuff. While you can easily accomplish this task when extracting proteins from mammalian cells (they have no cell walls to begin with), it can be more difficult when you are working with plants, yeast, bacteria, fungi and Archaea. These organisms have rigid cell walls that protect the basic cell structure against destructive mechanical forces.

Cell Walls 101
The unique composition of cell walls makes it difficult for researchers to extract their protein of interest. Most plant cell walls are usually composed of a primary and a secondary membrane. The primary membrane is made up of cellulose (a polysaccharide derived from glucose), pectin and hemicelluloses while the secondary membrane usually contains lignin, a complex organic molecule which makes up the wood of trees.

Algaecell walls are also made up of cellulose and other polysaccharides (mannan or xylan) while bacterial cell walls are composed of peptidoglycan (murein), a polymer consisting of sugars and amino acids that forms a strong mesh-like layer outside the plasma membrane.

The cell walls of most fungi are made up of chitin, the same material that forms the exoskeleton of arthropods while Archaea (single-celled microorganisms with no cell nucleus or any membrane-bound organelles in their cells) have cell walls made up of pseudopeptidoglycan, a polysaccharide that closely resembles peptidoglycan.

Breaking Down the Walls: Some Special Considerations
To extract your protein of interest from your starting material, you need to break down the cell wall or cell membrane without compromising the integrity of your protein. In addition, you also need to take into account the downstream application for the extracted protein when choosing the most appropriate cell lysis method. While this may seem like a no-brainer, this can be an especially tall order since these two often run counter to each other.

So, what are the methods that can help you tear down these rigid cell walls and how do you choose one that will satisfy all the requirements mentioned earlier? Here are some things you need to consider.

Mechanical methods involve sheer force in cracking the cell walls open. They do not require any reagents that may interfere with your protein of interest.

• Mortar and pestle technique. This method uses liquid nitrogen in grinding the sample and is considered as the fastest and most efficient way of extracting plant proteins and DNA. However, this method can be messy and physically demanding. It also poses a significant risk due to the amount of liquid nitrogen used.
• Beadbeating. This method can be used for any type of cell and is best used when working with large volumes of sample. However, since the apparatus can be very expensive, not everyone can afford to use this method.
• Sonication. This uses high intensity sound waves to disrupt cells. Its ease of use and adaptability to varying sample volumes makes it extremely popular but it also produces a lot of noise and can be extremely time-consuming. Overheating may also occur that damages or denatures proteins.
• Homogenization. This method is performed by squeezing cells through a tube to shear the outer layer (French Press) or by using a rotor-stator processor. Ideally used for small volumes and cultured cells. No an ideal technique for rigid cell walls.
• Freeze-thaw technique. The samples are frozen in a dry ice and/or ethanol bath or freezer and then thawed at room temperature or 37°C. The cells swell as ice crystals form during the freezing process. The thawing process causes the ice crystals to contract and ultimately break. 
• Use of high temperatures. The use of microwave or autoclaving quickly disrupts bonds within cell walls. However, this method can denature proteins so it should not be considered for samples that are sensitive to heat.

Non-mechanical methods use a combination of chemicals and mechanical force to break down cell wall components.

• Naturally occurring enzymes (cellulases, chitinase and/or bacteriolytic enzymes) can be used to remove the cell wall. 
• Organic solvents (alcohols, ether or chloroform) are often used in combination with shearing forces to increase the permeability of cell walls and membranes. This method is extremely helpful in extract hydrophobic molecules such as plant pigments.