Selecting the right protein purification method is important in achieving high yield and purity. The main aim of protein purification is to separate a single target protein from contaminating proteins to help investigate its structure and function. Protein purification is a key component of studying proteins, nucleic acids, and peptides for various purposes like clinical, industry, and research applications. However, determining the right protein purification system can present challenges, especially for scientists who are new to Protein purification procedures. Luckily, there are tips that can help one choose the best protein purification technique for their specific needs. Read on to learn more.
Protein purification is a series of processes made to isolate proteins from complex mixtures. This process is mainly used to analyze the structure, and function of proteins. Protein purification is a very crucial stage in biochemical and biomedical research since it makes it possible to isolate, characterize, and use proteins for a variety of scientific and industrial uses.
Getting pure samples of protein: Pure protein samples are needed to accurately research the proteins’ structure, function and protein-protein interactions. This minimizes the interference of contaminants on the outcomes of the experiments.
Determining the protein structure: Protein purification is an important step in determining the structure of proteins. It mainly involves isolating a protein from complex mixtures.
Functional studies: Purified proteins help in understanding their biochemical characteristics, such as enzymatic activity, regulatory mechanisms, and binding affinities. All these must be understood in order to piece together biological processes.
Quality control: Protein purification guarantees the integrity and purity of desired proteins by eliminating contaminants and impurities, which is essential for downstream applications’ appropriateness and the repeatability of experimental outcomes.
Biotechnological applications: The creation of biopharmaceuticals, diagnostic tests, and drug development are just a few of the biotechnological uses for purified proteins. It takes very pure proteins to create vaccines and therapeutic medicines.
Helps in disease research: The pathophysiology of different diseases, the identification of drug targets, and the discovery of biomarkers are all made easier through protein purifications. This information is helpful in the development of therapeutic alternatives for a number of diseases, such as cancer and neurological disorders.
First, you need to find out how your protein is expressed and where it comes from. You would need to use several expression systems, such as bacteria, insect cells, yeast, mammalian cells, or plants, depending on the kind and complexity of your protein. Every system comes with its pros and cons, including compatibility with the type of protein you have, yield, cost, scalability, and post-translational changes. It is imperative to take into account the method of tagging or labelling your protein since it may have an impact on its purification and characterization.
For instance, affinity tags like His and GST can help your protein attach more easily to particular resins or beads. As an alternative, you can enable the detection of your protein by fluorescence or luminescence by using fusion tags, such as GFP or mCherry.
Another thing to put in mind when selecting the method for protein purification is the quality and quantity of the protein. You might need a protein with a variable concentration and purity depending on your personal uses. For example, you may require a high level of purity and homogeneity if you are researching the protein structure or function of your protein.
However, if your protein serves as an enzyme or antigen, you can use a lesser degree of purity and concentration as long as it’s still active. It may be necessary to apply a variety of purification techniques, such as chromatography, dialysis, or precipitation, to get the required amount and quality of protein. Remember, each technique has its advantages and disadvantages.
Take into account the protein stability and solubility
The solubility and stability of your protein is a crucial consideration. You may need to adjust the settings and chemicals that can improve your protein’s stability and solubility based on its characteristics and interactions.
For instance, you might need to modify the protein solution’s pH, temperature, salt content, or buffer makeup. It might also be necessary to add stabilizers such as sugars, glycerol, polyols, or solubilizers like detergents or organic. These variables may have an impact on the quality, yield, reliability, and accuracy of protein purification. As such, you must thoroughly test and verify the ideal additives and optimal conditions of your protein.
You also need to take into account the function and activity of your protein when selecting the protein purification method. Here, you will have to apply several characterization techniques, like spectroscopy, mass spectrometry, or bioassays. This depends on your application goals and also research questions. The molecular weight, post-translational changes, amino acid sequence, conformation, binding, and catalysis of your protein are just a few of the details that each technique may reveal.
The Staley Lab at the University of Chicago faced challenges with traditional protein purification methods. The methods were time-consuming and led to suboptimal purity levels. Cody Hernandez, a PhD candidate in the lab, tried methods that can enhance the efficiency of protein purification to support his RNA splicing research. The lab adopted the CL7/Im7 Purification System, which enabled the use of a single-step purification procedure. This greatly reduced the time and effort needed to get high-purity proteins. The purification system utilized a novel affinity tag that binds to CL7 resin, allowing only selective capture of the target protein.
This case study shows how innovative approaches to protein purification can help overcome traditional challenges, which can, in turn, lead to more efficient research and better results.
Conclusion
This article has provided some helpful hints and recommendations for choosing the ideal protein purification and characterization technique for your biotechnology endeavour. Remember, there the perfect technique varies for each protein. To determine which approach works best for your protein, you might need to try a few different approaches and compare them.
When selecting a protein purification method, there are a number of things to take into account. Some of them are the protein’s properties, the intended use, the amount of protein required, and the purification process duration. A protein’s size, charge, solubility, and hydrophobic or hydrophilic nature are among its properties.
The protein quantity will also determine the kind of method to choose. Another consideration is the intended application of the protein, which will show the necessary level of purity. For example, some enzymatic assays need high levels of purity, while protein assays may require lower levels of purity.