What Is Protein Purification?

 

Protein purification is an important technique in biochemistry, molecular biology and biotechnology, as proteins play a vital role in various biological processes. Proteins are one of the important macromolecules and carry out a wide range of functions, such as catalyzing biochemical reactions, serving as structural components of cells and tissues and regulating gene expression.

The purification process involves several steps, including cell lysis, centrifugation, filtration, chromatography and electrophoresis. Each step helps separate different molecules depending on their physical and chemical properties, such as size, charge, hydrophobicity and affinity.

Protein purification can help study proteins’ structure, function and interactions. It also helps produce proteins for various applications, such as drug development, diagnostics and industrial biotechnology. Purified proteins also apply in various procedures, including crystallization, X-ray crystallography, NMR spectroscopy and other analytical techniques requiring highly pure and homogeneous samples.

This article will provide an overview of the various techniques used in protein purification, their principles, advantages and limitations. We will also look at the factors affecting the yield and purity of the purified proteins with the strategies for optimizing the purification process.

Additionally, we will highlight some of the applications of purified proteins in different fields and the challenges involved in scaling up protein production.

How does protein purification work?

Protein purification isolates and purifies a specific protein from a complex mixture of proteins in a biological sample. Protein purification involves cell lysis to separate the target protein from other contaminants, such as proteins, nucleic acids, lipids and carbohydrates.

Successfully separating proteins involves several protein purification steps, from sample preparation to protein isolation, purification, quantification and analysis.

Sample Preparation

The first step in protein purification is preparing the sample. It involves collecting the biological material containing the protein of interest, such as cells, tissues or fluids. The sample is then homogenized, breaking the cells or tissues to release the proteins. The homogenized sample is then centrifuged to separate the cellular debris and other unwanted materials from the protein-containing supernatant.

Protein Isolation

After the sample is prepared, the next step is isolating the protein of interest from the other proteins. Several methods are used for protein isolation, including chromatography, electrophoresis and precipitation. Chromatography is the most commonly used method, which involves the separation of proteins based on their size, charge, hydrophobicity or affinity for a specific ligand.

Protein Purification

Once the protein of interest is isolated, the next step is to purify it further to remove any remaining contaminants. This step involves additional chromatography techniques, such as ion exchange, size exclusion and affinity chromatography. The choice of chromatography technique depends on the characteristics of the protein, such as its charge, size and binding affinity.

Protein Quantification and Analysis

After the protein is purified, the final step is quantifying and analyzing it to determine its purity and concentration. Protein quantification involves determining the concentration of a specific protein in a given sample. This information is essential for many downstream molecular biology, biochemistry and biotechnology applications, such as protein-protein interactions, enzymatic assays and western blotting.

Several protein quantification methods exist, such as UV spectrophotometry, Bradford assay and bicinchoninic acid (BCA) assay. Each method has its advantages and disadvantages, depending on the specific requirements of the experiment.

UV spectrometry is a fast and simple method to measure the absorbance of the protein sample at 280 nm. The Bradford assay relies on the Coomassie Brilliant Blue dye binding to the amino acid residues of the protein and shifts its absorbance spectrum.

This method is more specific for proteins than the UV method. Finally, the BCA assay, the third method, utilizes a chelating agent, such as bicinchoninic acid, to bind to the copper ions in the presence of protein to form a purple-colored complex.

Protein analysis can be done using techniques such as SDS-PAGE, Western blotting and mass spectrometry, which can provide information on the protein’s molecular weight, structure and function.

Protein purification is a complex process requiring expertise in various techniques and methods. However, it is essential for studying the structure, function and interactions of proteins and developing protein-based therapeutics and diagnostics.

Protein purification techniques

Protein purification techniques are essential in many research fields, including biochemistry, molecular biology and biotechnology. Some protein purification techniques include:

Size Exclusion Chromatography

Size exclusion chromatography separates proteins based on size. Also known as gel-filtration chromatography, the technique involves a mixture of proteins through a column packed with a porous gel matrix.

The gel matrix contains pores of different sizes, allowing proteins of different molecular weights to enter and exit the matrix at different rates. Smaller proteins can enter the pores and take longer to exit the column, while larger proteins are excluded and eluted in this process.

This protein purification method is commonly used to separate proteins from large molecules, like nucleic acids, lipids and carbohydrates. Additionally, it removes aggregates, proteases and endotoxins from protein samples.

Ion-Exchange Chromatography

Ion-exchange chromatography is a technique to separate proteins based on their charge. This technique passes a protein mixture through a column packed with an ion-exchange resin.

The resin contains charged functional groups that attract or repel proteins based on their charge. The positively charged proteins bind to a negatively charged resin, while negatively charged proteins bind to a positively charged resin.

After binding, proteins are selectively eluted from the column by altering the pH or salt concentration of the buffer. This protein purification technique is useful for separating proteins with similar sizes but different charges. It is also useful for purifying proteins from complex mixtures such as cell lysates or serum.

Affinity Chromatography

Affinity chromatography involves separating proteins based on their specific interactions with ligands. This technique passes a protein mixture through a column packed with a resin containing a ligand that binds to the target protein. The target protein selectively binds to the resin while other proteins flow through the column.

After binding, the target protein is selectively eluted from the column by changing the buffer’s pH, temperature or salt concentration. This protein purification chromatography is useful for purifying high-specificity and yield proteins.

Hydrophobic Interaction Chromatography

Hydrophobicity is the tendency of a molecule or even a part of it to repel water. In other words, it refers to the relative insolubility of nonpolar substances in water. The hydrophobic interaction chromatography technique helps separate proteins based on their hydrophobicity. It passes a protein mixture through a column packed with a resin containing a hydrophobic ligand. The target protein will bind to the resin based on its hydrophobicity, while other proteins will flow through the column.

After binding, the target protein can be selectively eluted from the column by decreasing the salt concentration of the buffer. This protein purification chromatography is useful for purifying proteins with similar sizes but different hydrophobic properties.

Reverse-Phase Chromatography

This chromatography technique is used for protein purification based on the molecule’s hydrophobicity, similar to hydrophobic interaction chromatography. The technique uses a column packed with a hydrophobic resin, and the protein sample is loaded onto the top of the column.

The protein’s hydrophobic regions interact with the hydrophobic resin, causing the protein to bind to the column. The protein can be eluted from the column using a buffer with an increasing concentration of an organic solvent or by changing the pH.

Immunoaffinity Chromatography

Immunoaffinity chromatography is used to purify protein based on the interaction between an antibody and its antigen. The technique involves placing a column with a resin containing an antibody specific to the protein of interest.

The protein sample is loaded onto the top of the column, and the protein binds to the antibody. The other proteins in the sample are washed out, and the protein of interest is eluted from the column using a buffer that disrupts the interaction between the protein and the antibody.

Immunoaffinity chromatography is particularly useful when the target protein is at low concentrations. Alternatively, it is also beneficial when the protein has a specific post-translational modification or conformation recognized by the antibody.

The importance of protein purification

Proteins are complex molecules carrying a wide range of cell functions. However, the presence of impurities can affect their properties. Therefore, protein purification is essential for accurately characterizing and understanding t properties of the protein.

Obtaining a Pure Sample of Protein

One of the primary reasons for protein expression and purification is to obtain a pure sample of the protein of interest. A pure sample is essential for many downstream applications, such as structural studies, enzyme assays and protein-protein interaction studies. Contaminants in the sample can interfere with these experiments, leading to incorrect conclusions.

Determination of Protein’s Chemical Properties

Purification helps determine the protein’s precise molecular weight and amino acid sequence. This information helps identify and understand the protein’s structure and function. Further, protein purification can remove any post-translational modifications that may be present, allowing researchers to study the unmodified protein’s properties.

Studying Protein-Protein Interactions

Protein purification also helps study protein-protein interactions. The interaction between two proteins can be disrupted or inhibited by impurities in the sample, leading to inaccurate results. However, researchers can isolate the interaction between the two proteins of interest by purifying the proteins and accurately determining the binding affinity and specificity.

Helps Produce Recombinant Proteins

Protein purification is vital, as it helps produce recombinant proteins for therapeutic and industrial purposes. Impurities in the protein sample can affect safety and efficacy. As a result, purification is essential for ensuring the product’s purity and quality.

To sum up, protein purification helps study proteins and their functions. It accurately characterizes proteins and helps determine their precise properties and the isolation of protein-protein interactions. Purification is essential for downstream applications and producing high-quality therapeutic and industrial proteins.

Applications of protein purification

Protein purification is a process that separates and isolates a particular protein from a mixture of other proteins or cellular components. The purified protein can be used for various applications, including research, diagnosis and therapeutics.

Research

One of the primary uses of protein purification is in basic research. Researchers can isolate specific proteins to study their structure, function and interactions with other molecules. This information can provide valuable insights into biological processes and help researchers develop new disease treatments.

Therapeutics in Medicine

Purified proteins are often used as therapeutics in medicine. For instance, insulin, a hormone that regulates blood sugar levels, can be purified and used to treat patients with diabetes. Other purified proteins, such as growth factors and cytokines, have also been developed as drugs to treat various conditions, including cancer and autoimmune disorders.

Biotechnology

Protein purification is also used in the biotechnology industry to produce large quantities of specific proteins commercially. For example, enzymes used in food processing, such as rennet for cheese-making, can be purified and produced on a large scale.

Diagnostics

Protein purification is also important in diagnostics. Purified proteins can be used as diagnostic markers for specific diseases. For example, antibodies that recognize specific proteins associated with a particular disease can be purified and used in diagnostic tests to detect the disease.

What to consider for optimal protein purification results

Optimal protein purification results are essential for downstream applications, such as structural and functional analyses and therapeutic purposes. Here are some tips and considerations to ensure the best possible outcome:

Choosing the Right Protein Purification Method

Several protein purification methods are available, such as chromatography, precipitation and electrophoresis. Choosing the right method depends on the protein’s properties, size and purity required.

Preparation of Samples

It’s important to prepare the sample correctly before purification. It includes the removal of any contaminants, such as nucleic acids, lipids and other proteins, and adjusting the pH and salt concentration.

Optimization of Buffer Conditions

The buffer conditions, such as pH, salt concentration and ionic strength, can affect protein stability and solubility. Optimizing these conditions can increase the yield and purity of the protein.

Use of Appropriate Tags

Protein tags can aid in purification by allowing selective binding to specific resins or matrices. Choosing the appropriate tag can increase the yield and specificity of protein purification.

Monitoring Protein Purity

Various methods, such as SDS-PAGE, western blotting and ELISA, can assess protein purity. Regular monitoring of protein purity throughout the purification process can ensure the desired purity is achieved.

Storage and Handling of Purified Protein

Proper storage and handling can affect its stability and activity. Storage at the appropriate temperature, pH and buffer conditions can increase the protein’s shelf life and maintain its activity.

Quality Control

Quality control is crucial to ensure the purified protein meets the desired specifications. It includes assessing the protein’s identity, purity, activity and stability.

If you want optimal purification results, Avantor is here to help with its protein purification expertise. Our experienced professionals can guide you through every process step, from selecting the best purification method to optimizing your yield and purity. Contact us today to learn more and start your journey toward revolutionizing the healthcare industry.