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Read this article to learn about the meaning, principle strategies, selection and combination of purification techniques, purification of a tagged protein, evaluation of purification yield, concentration of purified protein and analysis of isolated proteins.
Introduction:
Protein purification is a series of processes intended to isolate a single type of protein from a complex mixture.
Protein purification is vital for the characterization of the function, structure and interactions of the protein of interest. The starting material is usually a biological tissue or a microbial culture.
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The various steps in the purification process may free the protein from a matrix that confines it, separate the protein and non-protein parts of the mixture, and finally separate the desired protein from all other proteins. Separation of one protein from all others is typically the most laborious aspect of protein purification. Separation steps exploit differences in protein size, physicochemical properties and binding affinity.
Purification may be preparative or analytical. Preparative purifications aim to produce a relatively large quantity of purified proteins for subsequent use. Examples include the preparation of commercial products such as enzymes (e.g., lactase), nutritional proteins (e.g., soy protein isolate), and certain biopharmaceuticals (e.g., insulin).
Analytical purification produces a relatively small amount of protein for a variety of research or analytical purposes, including identification, quantification, and studies of the protein’s structure, post-translational modifications and function. Among the first purified proteins were urease and Concanavalin-A.
Principle Strategies:
Most purification protocols require more than one step to achieve desired level of purity. Each step in the process will cause some loss of product, a yield of 80% in each step is assumed, and therefore, it is advisable to have as few purification steps as possible. Choice of a starting material is key to the design of a purification process.
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In a plant or animal, a particular protein usually is not distributed homogeneously throughout the body; different organs or tissues have higher or lower concentrations of the protein. Use of only the tissues or organs with the highest concentration decreases the volumes needed to produce a given amount of purified protein.
If the protein is present in low abundance, or if it has a high value, scientists may use recombinant DNA technology to develop cells that will produce large quantities of the desired protein (this is known as an expression system) Recombinant expression allows the protein to be tagged, e.g., by a His-tag, to facilitate purification, which means that the purification can be done in fewer steps. In addition to this recombinant expression usually starts with a higher fraction of the desired protein than is present in a natural source.
With background information, assays and sample procedures in place the three phase purification strategies can be considered. The purification has three phases of capture, intermediate purification and polishing, each with specific objective. In capture phase the objectives are to isolate, concentrate and stabilize the target procedure.
During intermediate purification phase the objective is to remove bulk impurities such as other proteins, nucleic acids, endotoxins and viruses. In polishing phase the objective is to remove any trace impurities and closely related substances. Therefore selection and optimum combination of purification techniques for capture, intermediate purification and polishing is necessary to ensure good yield and pure product.
The final purification process ideally consists of sample preparation, including extraction and clarification when required followed by above described three phases of purification. The number of steps will always depend on purity required and intended use of protein.
An analytical purification generally utilizes three properties to separate proteins. First, proteins- may be purified according to their isolectric points by running them through a pH graded gel or an ion exchange column. Second, proteins can be separated according to their size or molecular weight via size exclusion chromatography or by SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) analysis.
Proteins are often purified by using 2D-PAGE and are then analysed by peptide mass fingerprinting to establish the protein identity. This is very useful for scientific purposes and the detection limits for protein are nowadays very low and Nano gram amounts of protein are sufficient for their analysis.
Selection and Combination of Purification Techniques:
The aim of this combination is to evolve a fastest route to a product of required purity. For any chromatographic separation each different technique will offer a different performance with respect to recovery, resolution, speed and capacity. A technique can be optimized to focus on one of these parameters; for example, resolution to achieve the best between two parameters such as speed and capacity.
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A separation optimized for one of these parameters will produce result; quite different in appearance from those produced using the same technique but focused on alternative parameters. Therefore, it is always preferable to select a technique to meet the objectives in purification step.
Capacity in the simple model shown represents the amount of target protein loaded during the purification. In some cases the amount which can be loaded may be limited by volume (as in gel filtration) or by large volume of contaminants rather than the amount of target proteins.
Speed is of highest importance at the beginning where contaminants like proteases must be removed as quickly as possible. Recovery becomes increasingly important as the purification progresses because of the increased value of the purified product. The recovery’ is influenced by destructive processes in the samples and unfavorable conditions on the column.
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Resolution is achieved by the selectivity of the technique and efficiency of the chromatographic matrix to produce narrow peaks. In general, resolution is most difficult to achieve in final phases of purification where target protein and impurities have very similar properties.
Every technique offers a balance between capacity, speed, resolution and recovery and should be selected to meet the objectives of each purification step. In general, the optimization of any of these parameters can be achieved only at the expense of other and purification step will be a compromise.
The importance of each parameter will vary depending upon whether a purification step is used for capture, intermediate purification or polishing. This will steer the optimization of critical parameters as well as for the selection of suitable media for the step.
Purification of a Tagged Protein:
Adding a tag to the protein gives the protein a binding affinity it would not otherwise have. Usually the recombinant protein is the only protein in the mixture with this affinity, which aids in separation. The most common tag is the Histidine-tag (His-tag) that has affinity towards nickel or cobalt ions. Thus by immobilizing nickel or cobalt ions on a resin, an affinity support that specifically binds to histidine tagged proteins can be created. Since the protein is the only component with a His-tag, all other proteins will pass through the column, and leave the His-tagged protein bound to the resin.
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The protein is released from the column in a process called elution, which in this case involves adding imidazole, to compete with the His-tags for nickel binding, as it has a ring structure similar to histidine. The protein of interest is now the only protein component in the eluted mixture, and can easily be separated from any minor unwanted contaminants by a second step of purification, such as size exclusion chromatography or RP-HPLC.
Another way to tag proteins is to add an antigen peptide to the protein, and then purify the protein on a column containing immobilized antibody. This generates a very specific interaction usually only binding the desired protein. When the tags are not needed anymore, they can be cleaved off by a protease. This often involves engineering a protease cleavage site between the tag and the protein.
Evaluating Purification Yield:
The most general method to monitor the purification process is by running a SDS PAGE, of the different steps. This method only gives a rough measure of the amounts of different proteins in the mixture, and it is not able to distinguish between proteins with similar molecular weight.
If the protein has a distinguishing spectroscopic feature or an enzymatic activity, this property can be used to detect and quantify the specific protein, and thus to select the fractions of the separation, that contains the protein. If antibodies against the protein are available, then western blotting and ELISA can specifically detect and quantify the amount of desired protein. Some proteins function as receptors and can be detected during purification steps by a ligand binding assay, often using a radioactive ligand.
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In order to evaluate the process of multistep purification, the amounts of the specific protein have to be compared to the amount of total protein. The latter can be determined by the Bradford total protein assay or by absorbance of light at 280 nm, however some reagents used during the purification process may interfere with the quantification.
For example, imidazole (commonly used for purification of polyhistidine-tagged recombinant proteins) is an amino acid analogue and at low concentrations will interfere with the bicinchoninic acid (BCA) assay for total protein quantification. Impurities in low-grade imidazole will also absorb at 280 nm, resulting in an inaccurate reading of protein concentration from UV absorbance.
Concentration of the Purified Protein:
At the end of protein purification, the protein often has to be concentrated.
Different method exist for this purpose is:
1. Lyophilization:
If the solution does not contain any other soluble component than the protein in question the protein can be lyophilized (dried). This is commonly done after a HPLC run. This simply removes all volatile components leaving the proteins behind.
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2. Ultrafiltration:
Ultrafiltration concentrates a protein solution using selective permeable membranes. The function of the membrane is to let the water and small molecules pass through while retaining tin- protein. The solution is forced against the membrane by mechanical pump or gas pressure or centrifugation.
Analysis of Isolated Proteins:
This analysis is done by following techniques:
1. Denaturing-Condition Electrophoresis:
Gel electrophoresis is a common laboratory technique that can be used both as preparative and analytical methods. The principle of electrophoresis relies on the movement of a charged ion in an electric field. In practice, the proteins are denatured in a solution containing a detergent (SDS). In these conditions, the proteins are unfolded and coated with negatively charged detergent molecules. The proteins in SDS-PAGE are separated on the sole basis of their size.
In analytical methods, the protein migrates as bands based on size. Each band can be detected using stains such as Coomassie blue dye or silver stain. Preparative methods to purify large amounts of protein require the extraction of the protein from the electrophoretic gel. This extraction may involve excision of the gel containing a band, or eluting the band directly off the gel as it runs off the end of the gel.
In the context of a purification strategy, denaturing condition electrophoresis provides an improved resolution over size exclusion chromatography, but does not scale to large quantity of proteins in a sample as well as the late chromatography columns.
2. Non-Denaturing-Condition Electrophoresis:
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An important non denaturing electrophoretic procedure for isolating bioactive metalloproteins in complex protein mixtures is termed ‘quantitative native continuous polyacrylamide gel electrophoresis’ (QNC-PAGE).
