With the rapid advancement of molecular biology, especially in the field of genetic engineering, an increasing number of protein-based drugs have been developed and applied in the treatment of various diseases. As a result, the separation and purification of target proteins has become one of the fastest and most effective methods in biotechnology. This article provides an overview of the extraction, isolation, and purification processes of proteins.
First, the extraction of proteins (including enzymes) is a crucial initial step. Most proteins are soluble in water, dilute salt solutions, or buffers, while some, particularly those associated with lipids, can be extracted using organic solvents such as ethanol, acetone, or butanol. The choice of solvent depends on the nature of the protein and its solubility characteristics.
Aqueous solution extraction is the most commonly used method due to its ability to maintain protein stability and solubility. Typically, the volume of the solvent is 1–5 times that of the raw material, and gentle stirring is required to ensure complete dissolution. The temperature during extraction is usually kept low (below 5°C) to prevent denaturation, although some proteins may benefit from slightly higher temperatures for faster dissolution. To protect the protein from degradation, protease inhibitors like diisopropyl fluorophosphate or iodoacetic acid may be added.
The pH and salt concentration of the extraction buffer play a key role in the process. Proteins are ampholytes with an isoelectric point, so the pH of the solution should be adjusted to avoid this point to maximize solubility. Acidic proteins are often extracted in basic solutions, and vice versa. A small amount of neutral salt, such as NaCl at 0.15 mol/L, is typically added to enhance solubility without causing denaturation. Phosphate or carbonate buffers are frequently used to maintain a stable environment.
For proteins that are not easily dissolved in aqueous solutions, organic solvents like ethanol, acetone, or butanol can be employed. Butanol, in particular, is effective for extracting lipid-bound proteins due to its strong lipophilicity and mild denaturing effect. It is also suitable for a wide range of pH and temperature conditions, making it ideal for both animal and microbial sources.
Once extracted, proteins must be separated and purified. Several techniques are commonly used:
- **Salting out** involves adding neutral salts like ammonium sulfate to reduce protein solubility and cause precipitation. This method is widely used due to its efficiency and minimal impact on protein structure.
- **Isoelectric precipitation** takes advantage of the fact that proteins have minimal solubility at their isoelectric point, though it is often combined with other methods for better results.
- **Low-temperature organic solvent precipitation** reduces protein solubility by using methanol, ethanol, or acetone, offering high resolution but requiring careful control to avoid denaturation.
Other methods include **dialysis and ultrafiltration**, which separate proteins based on molecular size, and **gel filtration chromatography**, which separates proteins according to their size and shape.
**Electrophoresis** and **ion exchange chromatography** rely on the charge properties of proteins, while **affinity chromatography** uses specific ligands to bind and isolate target proteins efficiently. This technique is highly selective and can purify a single protein from a complex mixture in just one step.
Additionally, **hydrophobic interaction chromatography** exploits differences in the hydrophobicity of protein surfaces, and **genetic engineering-based purification markers**, such as GST, Protein A, and His-tagged systems, allow for targeted purification of recombinant proteins.
In conclusion, protein separation and purification are essential in biochemical research and drug development. Due to the complexity of biological systems, no single method is sufficient, and a combination of techniques is often necessary. Each step must be carefully optimized to ensure the integrity and functionality of the final product.
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