How to establish a protein purification process?

When establishing a protein purification process, the most important thing is to consider that the purified protein should be able to meet its subsequent application requirements. The quantity and purity of the protein must meet the requirements of experimental analysis. Moreover, because follow-up studies require active proteins that maintain a well-folded structure, information about protein behavior also needs to be considered. In the process of purification and subsequent storage, many treatment methods will affect the properties of the protein, such as unfolding, aggregation, degradation and inactivation of the protein. Make a detailed plan, complete the purification of the protein in the shortest time, and store it under the most stable conditions. It is considered that the entire purification process has been successfully completed.

Buffer system

In each step of the purification process, the solution environment of the protein is very important to maintain its stability and activity. The protein should be stored in a good buffer environment, and sudden pH changes should be avoided to prevent irreversible effects on the folding state, solubility and activity of the protein.

The buffer is an aqueous solution containing conjugated acid/base pairs. The pH range of the buffer is determined by its pKa value. The pKa value is defined as the pH value when 50% of the molecules have an acid structure and 50% have a basic structure (Figure 1). A general principle about buffers is that the pH value should be within 1 pH unit around the pKa value, so as to ensure that it has a good buffering capacity. This can ensure that there are enough molecules with acidic and basic structures that can be neutralized when H+ or OH- is added. In this way, the buffer can prevent changes in protein stability caused by changes in pH.

A good buffer must have the following characteristics:

• Water soluble

• Chemical stability

• Good buffering capacity in the required pH range

• Good compatibility with analysis and experimental application conditions

• Good compatibility with other solutes

Many substances can be used in biological buffers. The most commonly used buffer components usually have a pKa value close to neutral and can be used around the physiological pH range. Table 1 lists the 4 most commonly used biological buffers, their respective pH application ranges, and their advantages and disadvantages that may affect the protein purification process. To ensure sufficient buffer capacity, the concentration of these buffers is usually 25 mM.

Solution additives

In addition to a suitable buffer system, the solution used in the protein purification process-from lysis to storage-usually contains many other components that have a certain effect on the purity, stability and activity of the protein.

Protease inhibitors are often added in the lysis buffer and the early steps of the purification process to prevent the target protein from being digested by endogenous proteases. In the later stage of the purification process, these protease inhibitors are generally not added, because at this time almost all the proteases have been separated from the target protein. Metal chelating agents such as EDTA or EGTA are usually added to the protein storage buffer. These metal chelating agents bind to Mg2+ to prevent the target protein from being decomposed by the contained metalloprotease. There are some other additives, mainly used to protect the protein from being damaged and enhance its solubility.

Additives are used only when necessary. It may take multiple attempts to determine whether certain additives are effective for certain protein purification processes.

The influence of the expression system

The initial sample should be prepared before starting to purify the protein. Before the actual operation of protein purification, the first consideration is the source of the target protein. The source of the sample can be an original sample, such as liver, muscle, or brain tissue. Although researchers in the field of postgenomics rarely use purified proteins from original tissues, when researchers want to correlate the catalytic activity of a certain protein sequence, there is such a need.

Currently, it is more common to purify proteins from recombinant sources. Some important decisions need to be considered in advance to optimize subsequent purification. Researchers need to consider the end use of the protein (such as enzyme determination, structural research, antibody production), because this will determine the amount and purity of the final protein preparation. Although the detailed discussion of protein expression is beyond the scope of this article, there are still several basic points worth considering at this point because they directly affect subsequent protein purification.

Which system has the highest level of expression? The general rule is that the higher the expression level, the easier it is to obtain a large amount of highly purified protein.

1. If the E. coli expression system is selected, is the final goal soluble expression or insoluble expression in the form of inclusion bodies? Due to the high abundance of recombinant proteins in inclusion bodies, the isolation of inclusion bodies constitutes an important step in purification; this must be balanced with the difficulty of solubilization and the difficulty of refolding the target protein in the inclusion bodies and the final yield of soluble protein. . A lot of work has been done to optimize the yield of functional protein from inclusion bodies.

2. Another consideration is whether to target intracellular or extracellular (secreted) expression. Intracellular expression requires the purification of proteins from a large number of host cell proteins. In contrast, especially when the host cell is grown under serum-free conditions, the effective secretion of the target protein requires that the protein must be purified from a small amount of secreted host protein.

Regardless of the source of the target protein, as the initial step of purification, the preparation of the original sample is very important. At the same time, expression and purification strategies need to be considered.

Sample preparation

The extracellular secretion of the target protein can use a simple and rapid affinity purification protocol; the only sample preparation required may require the pouring of conditioned medium of adherent cells or low-speed centrifugation to remove suspended cells. Of course, the first step of the preparation process may require the addition of protease inhibitors or pH adjustment during chromatography, but these steps are simple and easy to implement. However, if ion exchange is performed in the first purification step, the sample may need to be desalted first. There may be technical difficulties when handling large amounts of conditioned media; dialysis or cross-flow filtration can often be used depending on the desalting volume of the media.

If the target protein is expressed intracellularly, the cells first need to be obtained by centrifugation, and then resuspended in a suitable lysis buffer. As mentioned above, the lysis buffer needs to contain suitable buffers and other additives to ensure maximum stability of the target protein. If the researcher avoids the time-consuming buffer exchange step before column chromatography, the composition of the sample/lysis buffer also needs to be compatible with the subsequent purification steps.

Next, an effective cell lysis method is needed. Various methods have been described in the literature. E. coli can be lysed by a French press (although this method is not easy to scale), ultrasound or detergent-based lysis (there are many commercial lysis reagents). The detergent-based lysis efficiency can be improved by adding lysozyme to the lysis buffer. Detergent-based lysis is very gentle and does not cause significant shearing of bacterial DNA. Therefore, in order to reduce the viscosity of the sample to produce a sample with good flow characteristics, it is usually necessary to add DNase.

Mammalian and insect cells can also be lysed by ultrasound or detergent-based methods. If the nuclear membrane is significantly lysed, DNase treatment may be required to reduce the viscosity of the sample.

Once the cells (microorganisms/insects/mammals) are lysed, it is usually necessary to centrifuge to remove cell debris (usually at 4°C at 15000×g for 15 minutes to avoid clogging the column). The resulting supernatant can be used to purify the target protein by column chromatography.

Other factors

Other factors in the protein purification process will also affect the stability of the protein. The fewer protein processing steps, the better, and the purification process with the fewest steps in the shortest time can ensure the highest yield of active protein. Moreover, during the entire purification process, the protein is best kept at a low temperature. The general purification process is carried out at 4°C, because this temperature can not only reduce the enzymatic hydrolysis speed (in the case of protease), but also ensure the structural integrity of the protein.

1. The ability of development and operation personnel to master the above content will affect the process design and operation process.

2. The accuracy and stability of the equipment and instruments used in the process will also affect the final result.

3. The quality of the reagents and drugs used in the process will also cause deviations in the results.

4. In addition to temperature, environmental factors also affect the final result.