General Principles and Method Selection of Protein Purification

2021-08-06

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With the development of molecular biology, more and more scientific researchers have mastered various experimental techniques of molecular biology, and developed a complete set of kits to make gene cloning and expression easier and easier. However, the upstream work of molecular biology is often not the ultimate goal. The key to molecular cloning and expression is to obtain pure expression products to study their biological effects, or to produce large quantities of biological products that can be used for disease treatment. Compared with upstream work, the downstream work of molecular cloning is more difficult, and protein purification is very complicated. In addition to ensuring purity, protein products must also maintain their biological activity. The purification process must be able to produce the same quantity and quality of protein every time, with good repeatability. This requires the application of very adaptable methods rather than the best method to obtain pure protein to purify the protein. A good method under laboratory conditions may fail in large-scale production applications, because the latter requires scale and good repeatability in daily applications.

protein purification

A protein tag refers to a peptide or protein expressed by fusion with the target protein using DNA in vitro recombination technology for the target protein's expression, detection, tracing, and Purification. Glutathione S-transferase GST is a protein purification tag commonly used in laboratories, which is a transferase that plays a vital role in the detoxification process and can specifically bind to glutathione, showing the principle of enzyme and substrate action.

So using this principle can make GST into a tag to express a fusion protein that specifically binds to an affinity medium with a glutathione ligand, thus purifying the target protein and then using a specific enzyme to excise the GST tag to obtain the natural protein.

General principles of protein purification

Protein purification must use the inherent similarities and differences between different proteins, use the similarities between various proteins to remove the pollution of non-protein substances, and use the differences of each protein to purify the target protein from other proteins. Each protein has differences in size, shape, charge, hydrophobicity, solubility, and biological activity. Using these differences, the protein can be extracted from a mixture such as E. coli lysate to obtain a recombinant protein. Protein purification can be roughly divided into two stages: coarse separation stage and fine purification stage. The coarse separation stage mainly separates the target protein from other cellular components such as DNA and RNA. Due to the large sample size and mixed components, the resin used requires high capacity, high flow rate, large particles, and wide particle size distribution.

In order to quickly separate the protein from the contaminants, to prevent the target protein from being degraded. The fine purification stage requires higher resolution. In this stage, the target protein must be distinguished from those with similar size and physical and chemical properties. Smaller resin particles are used to improve the resolution of commonly used ion exchange columns and hydrophobic columns. Two factors, the selectivity of the resin and the efficiency of the column, should be considered comprehensively when applying. Selectivity refers to the specificity of the binding between the resin and the target protein, and the column efficiency refers to the ability of each component of the protein to be eluted from the resin one by one. The narrower the elution peak, the better the column efficiency. With only good selectivity, the elution peak is too wide, and the protein cannot be separated effectively.

GST-tag protein purification

The GST tag protein is generally used in prokaryotic expression systems because it is a highly soluble protein, and hopes that it can be used to increase the solubility of exogenous proteins; the other is that it can be expressed in large quantities in E. coli and play a role in increasing the expression.

The primary preparation process of GST tag is a complete process involving pre-sample expression and post-protein purification, as follows.

One,Selection of vector

The fusion protein with GST tag is constructed by inserting a specific gene or gene fragment into the polyclonal site region of the pGEX vector. The fusion protein is induced to be expressed by the lactose analog IPTG under the regulation of the tac promoter.

Selection requires attention.

(1) Select a vector that is consistent with the reading frame of the inserted fragment protein to avoid code shift mutations.

(2) Select the protease and enzymatic reaction conditions suitable for the target protein.

Two,Host

Although various E. coli can clone and express pGEX vectors, some specifically engineered strains can be more suitable for expressing full-length tag proteins with higher yields. Some cytoplasmic protease (Lon, OmpT, DegP, or HtpR) deficient strains can effectively protect the target protein from degradation by the host bacteria.

E. coli BL21, deficient in OmpT and Lon proteases, can express the protein at a higher level and is a recommended host strain for studies expressing GST-tagged proteins.

Due to the low transformation efficiency of BL21, other strains (e.g., JM105) can be used for cloning and amplifying vectors, but E. coli with recA1 alleles cannot amplify pGEX vectors.

In addition to E. coli, phage M13KO7 Helper Phage is the M14 phage that can be used for the GST expression system.

Three,Insert fragment DNA

The coding region of the inserted DNA fragment cannot exceed 2kb, and the end of the inserted fragment must be guaranteed to be complementary to the linearized vector end. Directional cloning using different endonucleases allows the fragment to be inserted in the correct direction.

Four,Optimization of expression

The key to this step is to select the best protein expression level and corresponding growth conditions from multiple clones. After these conditions are determined, large-scale culture can be performed. Optimization of expression conditions includes medium, temperature, induction conditions, aeration, positive clone selection, inclusion control, etc., while low temperature, increased aeration, or different induction conditions are the first to be considered.

Five, Purification

GST fusion proteins can be easily purified from bacterial lysates by affinity chromatography.

Detection indicator

Several methods detect GST-tagged proteins, depending mainly on the experimental conditions. Among them, SDS-PAGE is the most commonly used method for detecting experimental results, but it is not recommended for detecting high-throughput screening samples.

Seven,Tag excision

If structural or functional studies are performed on the target protein, going out to the GST tag is often necessary. For the excision of the tag, either on-column or off-column can be chosen to be enzymatically cleaved in the eluted solution.

Protein purification labeling of GST in the pathogenesis of drug-related liver injury.

As the most significant metabolic organ and detoxification organ in the body, the liver is susceptible to damage by various drugs and metabolites entering the body. Drug-induced liver injury (DILI) is a disease caused by various chemical drugs, biological drugs, herbal medicines and their preparations, health products, dietary supplements, and metabolites that induce hepatocyte damage and necrosis, liver enzyme abnormalities, and related clinical manifestations.

Drug metabolizing enzymes are the main targets of molecular studies related to drug-related liver injury, and glutathione S-transferase (GST) is one of the essential metabolizing enzymes. GST has a high concentration in the hepatocyte solute and can reduce toxicity and promote urinary excretion by catalyzing the binding of glutathione (GSH) to active metabolites in the liver. When the liver is severely injured, GST and GSH will be released from the hepatocyte cytoplasm into the plasma, resulting in reduced GST activity in the hepatocyte cytoplasm. Therefore, gene regulation of GST is decisive for GSH metabolism, and clinical monitoring of GST activity levels in liver tissues may be a potential strategy for diagnosing DILI.