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Comprehensive Guidelines to Protein Purification: Methods, Techniques, and Applications
2024-10-30
What is Protein Purification?
Protein purification is the process of separating and purifying proteins from mixtures, and obtaining high-purity protein. Recombinant Protein, due to their different sizes, structures, amino acid sequences, can affect subsequent protein function determination and obtain high-purity pure proteins. In downstream applications such as protein function determination, protein protein interactions, etc., high-purity proteins have little impact on the results, while proteins with other impurities may lead to inaccurate experimental results. In addition, high-purity proteins help to accurately determine molecular weight and amino acid sequence, which is beneficial for structural research. In summary, protein purification plays a crucial role in the protein production process.
Protein Purification Protocol
Figure 1: Workflow protein purification. Source: Technology Networks.
To ensure high yield and purity of recombinant protein production, protein purification is crucial. If the purity is high but the yield is low, it will affect the next experiment, while high yield and low purity will produce many impurities other than the target protein, both of which will affect the experimental process. Therefore, it is necessary to strictly control the steps of protein purification. The following are the steps of protein purification:
Obtain Protein
Proteins include natural proteins isolated from tissues, cells, or the entire organism, as well as recombinant proteins produced through genetic engineering techniques.
Protein Extraction
Figure 2: Protein extraction methods. Source: Technology Networks.
Crushing cells containing the target protein, destroying the cells through repeated freezing and thawing, ultrasonic treatment, detergent, and other methods, and finally removing cell debris through differential centrifugation to allow the protein to be secreted into the surrounding solution. The specific methods of protein extraction are:
Mechanical crushing: including homogenization (mainly for tough plant and animal tissues), ultrasonic treatment (for bacteria), and pressure cycling (for yeast).
Chemical fragmentation: including detergents, organic solvents (ethanol or acetone), Chaotropical agents (urea and guanidine hydrochloride).
Repeated freeze-thaw cycles.
Enzymatic dissolution: Lysozyme (can only be used for bacterial cells).
Dissolution and Stability
By adding an appropriate amount of buffer or protease inhibitor to prevent protein degradation, it is also necessary to ensure the solubility of proteins in the solution and prevent protein aggregation.
Protein Purification
Methods of protein purification: chromatography (affinity chromatography, ion exchange chromatography, gel filtration chromatography, also known as size exclusion chromatography, high-performance liquid chromatography, hydrophobic interaction chromatography), precipitation (differential centrifugation to remove cell debris), ultrafiltration and dialysis (concentration and desalination, for the removal of small molecular impurities) and other methods to purify proteins.
Analysis and Characterization
Roughly estimating the content of different proteins through electrophoresis (SDS-PAGE) cannot distinguish proteins with similar molecular weights. If there are antibodies targeting the target protein, quantitative detection of the protein can be achieved through WB and ELISA. If there are unique optical properties, the structure and concentration of proteins can be determined by spectroscopy. Protein activity and total yield can be determined through SPR. Advanced technologies such as nuclear magnetic resonance spectroscopy, X-ray, and mass spectrometry can also be used to determine proteins.
And each purification technology has its own scope of application and advantages and disadvantages:
Centrifugation is suitable for crude sample extraction, which is convenient and efficient, but cannot remove small pollutants.
Precipitation is suitable for fractionation, which is fast and simple, but the target product co precipitates with pollutants and requires further processing.
Affinity chromatography is suitable for labeling proteins and has affinity, with the advantage of high specificity and purity. However, resin prices are expensive and ligand binding needs to be optimized.
Ion exchange chromatography requires the protein to have a clear charge, and attention should be paid to the pH value of the solution.
Gel filtration chromatography (size exclusion chromatography) can separate proteins with different molecular weights. The purification process is mild and not easy to destroy proteins.
Hydrophobic interaction chromatography requires proteins to have different hydrophobicity in order to purify proteins using this method, but it requires salt solution optimization, which may cause damage to the proteins.
Different methods need to be selected based on the sample and applicable scope.
Figure 3: Chromatography techniques. Source: Technology Networks. 
Recombinant Protein Production
Recombinant protein production is a biotechnology that introduces recombinant DNA into expressing host cells to produce proteins in high yields. Common host cells include bacteria (Escherichia coli, Bacillus subtilis), yeast (Saccharomyces cerevisiae, Pichia pastoris), insects, mammals, etc.
1. Gene cloning
After identifying the target gene, the target gene is inserted into a plasmid expression vector through endonuclease and DNAligase using recombinant DNA technology.
2. Transformation and transfection
Inserting recombinant DNA into host cells, including bacteria, yeast, insects, and mammalian cells, is called transformation for yeast and bacterial cells. During the transformation process, DNA is transferred from one bacteria to another and integrated into the recipient genome through homologous recombination. For insect cells and mammals cells, it is called transfection, which is the change in animal cell characteristics.
3. Screening
Through antibiotic screening, host cells carrying resistance marker genes in the vector will be identified.
4. Expression
Cultivate and screen host cells under suitable conditions such as temperature, pH value, and nutrient level, and optimize the conditions.
5. Protein production
Release protein and proceed with protein purification.
Figure 4: Recombinant protein expression protocol. Source: Technology Networks.
Recombinant Protein Purification
Recombinant protein purification requires the introduction of tags that promote dissolution to facilitate protein purification while maintaining the original function of the protein.
Common tags include: His-Tag GST-Tag, Strep-Tag, FLAG-Tag, GFP-Tag, MBP-Tag, Halo-Tag, CBP-Tag, Protein A/G Tag. The most commonly used are His Tag purification and GST Tag purification:
His Tag Purification
The His tag consists of six histidine residues located at the N-terminus or C-terminus of the protein. During protein purification, the affinity of the protein for metal ions is increased by adding the tag, which then binds to the carrier that fixes the metal ions. The protein with the tag added will bind to the carrier, while other impurities will be washed away. The substances remaining on the carrier will be separated by elution to isolate the target protein.
GST Tag Purification
A glutathione S-transferase that can bind to glutathione fixed on resin and subsequently elute with reduced glutathione to separate from other proteins or impurities.
| Tags Types | Function | Example |
|---|---|---|
| Affinity tags | Tags can be used as purification tags | CBP, MBP, Strep, GST |
| Dissolving tags | Tags that increase protein solubility | TRX, NANP, MBP, and GST dual solubilization |
| Chromatographic Tags | Change chromatographic characteristics | FLAG tag |
| Epitope Tags | Suitable for western blotting, immunofluorescence, and immunoprecipitation experiments. | Short peptide sequence ALFA , V5, Myc, HA, Spot, T7, and NE |
| Fluorescent Tags | Introducing Fluorescent Color | GFP |
Protein Function Assay
It is important to analyze and characterize purified proteins, which can usually be analyzed from aspects such as concentration, purity, activity, structure, stability, and interactions.
Protein Concentration Determination
Bradford assay: The Coomassie Brilliant Blue dye itself appears brownish red (with a maximum absorbance of 465 nm), but when bound to proteins in acidic environments, it turns blue (with a maximum absorbance of 610 nm) and is affected by detergents.
BCA assay: It can measure the total protein content. In an alkaline environment, the protein reduces Cu2+to Cu+, and Cu+reacts with BCA to form a complex that can absorb 562nm wavelength. It is measured by a spectrophotometer and forms a standard curve through BSA. The standard curve is used to measure the concentration of protein.
Determination of Protein Purity
SDS-PAGE: Sodium dodecyl sulfate (SDS, also known as sodium dodecyl sulfate) and polyacrylamide gel. Proteins can be separated by different sizes, and the measurable molecular weight is usually between 5-250kDa, unaffected by protein shape and charge.
Figure 5: SDS-PAGE. Source: Wikipedia.
Western Blot: separate proteins of different sizes through SDS-PAGE, transfer the target protein to polyvinylidene fluoride (PVDF), and specifically bind to the target protein through primary antibody. Then, add a secondary antibody with an enzyme label that binds to the primary antibody, and measure the protein by adding a substrate that can make the secondary antibody color.
Figure 6: Western blot protocol. Source: Wikipedia.
Enzyme linked immunosorbent assay (ELISA): the antigen of the test sample is attached to a solid support, and the first antibody is added to specifically bind to the coated antigen, washing away the unbound antibody. The first antibody contains an enzyme, and a color change is observed after adding a substrate that reacts with the enzyme.
Protein Structural Analysis
X-ray crystallography: X-ray crystallography can determine protein structure by the directional changes of X-rays, and is also used to determine the interactions between drugs and target proteins.
Protein-protein Interaction
Surface Plasmon Resonance (SPR): SPR can be used to detect interactions between molecules, if protein to protein binding leads to an increase in SPR signal.
Bio layer interferometry (BLI): BLI enables real-time analysis of biomolecule interactions without the need for fluorescent labeling. BLI can detect proteins, nucleic acids, polysaccharides, lipids, small molecule drugs, antibodies, viruses, bacteria and cells and other samples.
Yeast two hybrid system (Y2H): Yeast two hybrid screening can detect interactions between two proteins or between proteins and DNA, and can also provide yeast hybrid libraries for cells, tissues, or organisms.
Pull-down: Pull Down experiment, a technology to detect molecular interactions in vitro conditions, which is used to verify protein-protein interactions or to screen for target proteins.
Co-immunoprecipitation (COIP): Co-immunoprecipitation (Co-IP) technique is a classical method used to study protein-peptide and protein-protein interactions based on specific immunoreactivity between antibodies and antigens, and it is an effective method for determining the physiological interactions between proteins and peptides, and proteins and proteins in intact cells
Chromatin Immunoprecipitation (CHIP): Chromatin Immunoprecipitation (ChIP) is a powerful tool for studying protein-DNA interactions in vivo, and it can be used not only to detect the dynamic interactions between trans factors and DNA in vivo, but also to study various covalent modifications of histones as well as the relationship between transcription factors and gene expression.
Comprehensively review protein purification, which is an important step in the protein production process and provides key points for subsequent experiments and research.
ReferenceReference
[1] https://www.technologynetworks.com/tn/articles/an-introduction-to-protein-purification-methods-technologies-and-applications-388443
[2] Labrou, N. E. (2021). "Protein Purification Technologies". Protein Downstream Processing. Methods in Molecular Biology. Vol. 2178. pp. 3–10. doi:10.1007/978-1-0716-0775-6_1. ISBN 978-1-0716-0774-9. PMID 33128738. S2CID 226224596.
[3] Scopes RK (1994). Protein Purification - Springer. doi:10.1007/978-1-4757-2333-5. ISBN 978-1-4419-2833-7.