Protein Extraction/Purification Reagents

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What Is Protein Extraction/Purification?

In the typical protein extraction/purification process, there are three main steps:

1. Lysis/extraction of total protein from the sample
2. Separation and concentration of the target protein
3. Removal of interfering substances and impurities (cleanup of extracted proteins)

The first step, crushing/lysis of cells and extraction of proteins, is critical because it affects the yield and quality of the final protein obtained.^1）

3 points of Protein Extraction

The following three points must be considered for efficient recovery of the target protein:

1. Type of sample

Mammalian Tissue

When proteins are extracted from tissues, the tissues should be disrupted by ultrasound or a homogenizer.

Mammalian Cells, Tissue Culture Cells

The cell membrane (a lipid bilayer) needs to be disrupted in a solution containing a detergent or other agent to extract proteins.

E. coli, Yeast, Plants

E. coli, yeast, and plant cells all have cell walls. Physical or enzymatic disruption methods are used to break down the cell wall.

2. Localization of the Target Protein

Some proteins are localized in the endoplasmic reticulum, mitochondria, nucleus, or cell membrane. In the protein extraction process, non-target proteins are removed, and fractions that contain high levels of the target protein are selectively retained so that the target protein is recovered at high concentration. Protein extraction reagents from different cell fractions are commercially available.

3. Stabilization of the Protein During Extraction (Inhibition of Proteases)

The cell structure is disrupted during the protein extraction process, resulting in release of intracellular proteases. If untreated, the target protein is degraded by the proteases.

To prevent degradation, protease inhibitors are added to the extraction reagents in advance or to the protein extracts. Since many proteases are most active at around 37°C, the extraction procedure is performed while keeping the sample at a low temperature (0-4°C). In general, samples and reagents are placed on ice during the extraction process.

When the proteins of interest are phosphoproteins, phosphatase inhibitors are used in addition to protease inhibitors to prevent dephosphorylation of phosphoproteins.

Various Protein Extraction Methods

Disruption by Ultrasound

Tissues and cell structure are disrupted by pulses of high-frequency ultrasound, a process also called “sonication.”

A probe that emits ultrasonic waves is immersed in a sample suspension mixed with extraction reagents, and the sample is exposed to continuous impact of air bubbles generated/burst from the energy produced by the ultrasound. Since the process generates heat, ultrasound is applied intermittently while the sample suspension is cooled to prevent the temperature from rising.

Disruption by Homogenizers

Homogenizers (ex. BioMasher) are often used to disrupt tissues and tissue culture cells. A rod called a “pestle” is pressed against the sample to disrupt cells. The number and speed of pressing strokes affect the efficiency of disruption.

• Dounce homogenizer: round glass pestle + glass tube
• Potter homogenizer: round or conical Teflon-coated pestle + outer tube

Disruption by French Press

A cell suspension of about 50 to 200 mL is placed under high pressure and pushed through a small hole to disrupt the cells.

Disruption by Bead-Beating

Fine beads are used to disrupt the cell wall and membrane. This technique is often used for protein extraction from yeast cells, which have cell walls. The efficiency depends mainly on the mass and diameter of the beads. Appropriate beads should be selected for the sample.

Mortar and Pestle

The sample is disrupted by grinding with a mortar and pestle. In some cases, the sample is frozen in liquid nitrogen prior to grinding. This technique is often used to disrupt plant cells with rigid cell walls composed of cellulose and polysaccharides.

Freeze-Thaw Method

Samples are quick-frozen in liquid nitrogen, on dry ice, or in cold ethanol, and thawed at room temperature or 37°C. The freezing process causes the cells to expand and form ice crystals, which disrupt the cells for subsequent protein extraction. The process is usually repeated at least two or more times until sufficient disruption is achieved. The freeze-thaw method is used for protein extraction from bacteria and mammalian cells.

Enzymatic Digestion Method

Enzymes that degrade cell walls and other substances can be used for protein extraction. For example, lysozyme is used for E. coli, and cellulase and pectinase are used for plants.

Achromopeptidase^®, a peptidase with a broad bacteriolytic activity, is also used. It has a more potent bacteriolytic activity than lysozyme and works on a wide variety of bacteria.

Osmotic Shock Method

Proteins are extracted from cells placed in a solution with low osmolality, which induces an osmotic shock. The advantage of this method is that intracellular organelles remain intact, resulting in lower protease activities.

Detergent-based Methods

Detergents are amphipathic molecules consisting of a hydrophilic head group and a hydrophobic tail with aliphatic or aromatic properties. Detergents are used to increase the solubility of proteins with low affinity for water (high hydrophobicity). They can be added directly to the cell lysate. To purify membrane proteins, detergents are added after the membrane fraction is prepared. Detergents can be ionic (anionic or cationic), nonionic (uncharged), or amphoteric (having both positive and negative charges, with no net charge). Which detergent to use depends on the sample preparation procedure. Generally, nonionic or amphoteric detergents are often used. Digitonin is one of the commonly used detergents.

References

1. “Protein Experiment Notebook vol.1 4th ed.” ed. by Okada, M., Miyazaki, K., Yodosha, (2011). (Japanese)