Protein purification is an essential step in biological research, drug development, and biotechnology processes. By isolating specific proteins from mixtures, scientists can characterize their structure, function, and interactions with other molecules.
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Fig. 1 Basic steps of protein purification. (Du M, et al. 2022)
Methods of Protein Purification
Precipitation Techniques
One of the earliest methods for protein purification is precipitation. This technique relies on the ability to induce proteins to aggregate and separate from the solution. Common precipitating agents include salts, organic solvents, and polymers.
Chromatography
Chromatography has become the backbone of modern protein purification. This versatile technique separates proteins based on their physicochemical properties, such as size, charge, and affinity. There are various types of chromatography, including:
Ion-Exchange Chromatography: Separates proteins based on their net charge. Positively charged proteins bind to a negatively charged resin, while negatively charged proteins pass through.
Size-Exclusion Chromatography: Separates proteins by size, allowing smaller proteins to enter the porous matrix of the column and larger proteins to orbit the beads.
Affinity Chromatography: Exploits the specific binding between a protein of interest and a ligand immobilized on the column. This method is highly selective and can purify single proteins from complex mixtures.
High-Performance Liquid Chromatography (HPLC): HPLC is a high-performance chromatography technique that uses a high-pressure pump to push a liquid solvent containing the protein mixture through a column. It provides high resolution and sensitivity, making it a preferred method for purifying proteins with demanding purity requirements.
Ultrafiltration and Dialysis
Ultrafiltration and dialysis are membrane-based techniques based on the principle of size exclusion. Ultrafiltration separates proteins based on their molecular weight by passing them through a semipermeable membrane. Dialysis involves the diffusion of proteins across a membrane, usually against a concentration gradient, to remove unwanted small molecules.
Precise Fractionation
Fractionation involves separating proteins based on their isoelectric points (pI) using techniques such as isoelectric focusing (IEF). In IEF, proteins migrate to their pI under the influence of an electric field, resulting in distinct bands that can be harvested for further purification.
Technological Advances in Protein Purification
In recent years, protein purification technology has advanced significantly due to the need for higher throughput, higher purity, and cost-effective methods. A notable development is the advent of automated systems that streamline the purification process, reducing manual labor and enhancing reproducibility.
Magnetic Bead-Based Purification
Magnetic beads coated with ligands specific to the target protein have emerged as a powerful tool for protein purification. These beads can be easily manipulated using magnetic fields, simplifying the purification process and enabling automation.
Microfluidic-Based Purification
Microfluidic devices provide miniaturized platforms for protein purification. These systems manipulate small volumes of fluids, providing rapid and efficient purification with reduced reagent consumption.
Protein purification is a dynamic field that is constantly evolving with advances in technology and methodology. From early precipitation techniques to today's sophisticated automated systems and new technologies, scientists have made remarkable progress in unraveling the complexity of proteins. As our understanding of proteins deepens thanks to innovative purification methods, the door is wide open to new discoveries and applications in medicine, biotechnology, and more.
Reference
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Du M, Hou Z, Liu L, Xuan Y, Chen X, Fan L, Li Z, Xu B. 1Progress, applications, challenges and prospects of protein purification technology. Front Bioeng Biotechnol. 2022, 10:1028691.
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