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Introduction

• Protein purification is a collection of processes designed to extract and purify a specific protein or protein complex from cells, tissues, or entire organisms. The primary purpose of protein purification is to analyze the protein's function, structure, and interactions. These separation processes typically take advantage of differences in protein size, physicochemical properties, binding affinity, and biological activity.
• There are two main types of protein purification: preparative and analytical. Preparative purification focuses on generating a large quantity of purified proteins for practical applications, such as enzyme production (e.g., lactase), nutritional protein creation (e.g., soy protein isolate), and biopharmaceutical development (e.g., insulin). This type of purification involves multiple steps and rigorous quality control to remove any other host proteins or biomolecules that could pose a risk to human health.
• On the other hand, analytical purification aims to obtain a small amount of a protein for research or analytical purposes. These may include protein identification, structural analysis, and investigations into the protein's structure, post-translational modifications, and function.

Chromatographic methods

Chromatography is a crucial technique in protein purification methods, as it enables the isolation of a specific protein from a mixture containing thousands of different proteins found in cells and tissues. The separation of proteins in chromatography relies on the type of column used and the chemical or physical properties of the protein molecule. There are four primary chromatography types employed for protein separation:

• Size exclusion chromatography: This method separates proteins based on their size, shape, or molecular weight.
• Ion exchange chromatography: In this method, proteins are separated according to their charge or isoelectric point.
• Hydrophobic interaction chromatography: This technique is similar to reverse phase columns used for purifying organic molecules and separates proteins based on their relative hydrophobicity.
• Affinity chromatography: This method separates proteins based on their ability to bind to a specific ligand that is covalently attached to a column bead.

In preparative protein purification, the purification process typically consists of one or more chromatographic steps. The fundamental procedure in chromatography involves passing a solution containing the target protein through a column filled with a chromatography resin designed to separate proteins based on a particular property. Different proteins interact with the column material in varying ways, allowing separation based on the time it takes for the protein to pass through the column or the conditions needed to elute the protein from the column. As proteins elute from the column, they are usually detected by measuring their absorbance at 280 nm, which corresponds to the absorption of light by aromatic amino acids.

Electrophoresis

• Electrophoresis is a major biochemical method used for molecular separation. This technique can be one-dimensional or two-dimensional. One-dimensional electrophoresis is commonly used for standard protein and nucleic acid separations, while two-dimensional separation of proteins is employed for fingerprinting and can accurately resolve over 1,500 proteins present within a cell when properly executed.
• When proteins are used with the detergent SDS (sodium dodecyl sulfate), they become negatively charged due to their attachment to the SDS anions. The separation process that occurs on a polyacrylamide gel is known as SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis). This technique has become a standard method for determining molecular weight.
• In clinical testing for biochemical diseases, protein analysis is often preferred over gene examination. Many of these genetic diseases are referred to as "inborn errors of metabolism" because they are present at birth and interfere with crucial metabolic pathways. Depending on the specific disease, tests can be designed to directly measure protein activity, metabolite levels, or protein size/quantity. These tests typically require a tissue sample, such as blood, urine, amniotic fluid, or cerebrospinal fluid, in which the protein is present. Since gene products may degrade more rapidly than DNA or RNA, it is important to collect, store, and ship the sample promptly and according to the laboratory's guidelines.
• Various technologies, such as high-performance liquid chromatography (HPLC), gas chromatography/mass spectrometry (GC/MS), and tandem mass spectrometry (MS/MS), are used to detect and quantify metabolites. Moreover, bioassays may use fluorometric, radioisotopic, or thin-layer chromatography methods.

Conclusion

Protein purification is a crucial process in both practical applications and research. It involves the separation of specific proteins from complex mixtures using various techniques. Chromatography is the primary method for protein purification, with size exclusion, ion exchange, hydrophobic interaction, and affinity chromatography being the most commonly employed techniques. Additionally, electrophoresis, particularly SDS-PAGE, is a standard technique for determining molecular weight and analyzing proteins. In clinical testing for biochemical diseases, protein analysis techniques such as HPLC, GC/MS, and MS/MS are used to detect and quantify metabolites, while bioassays may involve fluorometric, radioisotopic, or thin-layer chromatography methods. Overall, protein purification and analysis are essential for understanding protein function, structure, and interactions in various biological systems.