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Protein expression and purification protocol

Update time : 2020-10-15

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Protein expression and purification protocol

The study of protein functions and the production of functional proteins are basically inseparable from the heterologous expression and purification of proteins. 

Today I will talk to you about protein expression and purification from the following aspects.

1. Label selection

Currently common expression tags are His-tag (histidine tag), GST-tag (glutathione sulfhydryl transferase tag), MBP-tag (maltose binding protein tag), CBD-tag (chitin binding region tag) )Wait. 

Each tag has unique properties, such as the molecular weight of the tag, its ability to regulate protein solubility, its effect on protein folding, whether it needs to be excised, etc. 

Choose a suitable label according to the characteristics of the protein you want to purify. The label is selected well, and subsequent experiments will get twice the result with half the effort.

2. Vector construction

After selecting the label to be used, it is necessary to prepare an expression vector. At present, there are many commercialized vectors to choose from, such as the pET28 series with His-tag and the pMAL series with MBP-tag. 

Most vector series include the form of inserting the tag into the N-terminal or C-terminal, which can be flexibly selected according to the characteristics of the protein. 

When inserting the protein coding gene, you need to pay attention to avoid frameshift. If you want to construct a vector from scratch, don’t forget to insert a suitable promoter and transcription termination sequence. Promoter sequences generally use inducible promoters, constitutive promoters will cause premature expression and accumulation of foreign proteins, which is not conducive to host proliferation.

3. Host selection

Common hosts are E. coli, mammalian cells, yeast, and insects. For E. coli, the most suitable strain for heterologous expression is BL21 and its derivative strains, but it still needs to be selected according to the transcription and translation elements selected in the vector. 

The endogenous protease gene in BL21 is missing, so it is a strain suitable for foreign protein expression, but it does not have T7 RNA polymerase, so it cannot be used for protein expression containing T7 promoter; BL21(DE3), in BL21 Based on the integration of the T7 phage genome, it is suitable for T7 expression systems, such as the pET series; BL21(DE3)PLySs, based on BL21(DE3), with the T7 lysozyme gene, which can more strictly control the expression of T7 polymerase. 

It should be noted that due to the existence of genes such as endonuclease (endA1) recombinase (recA1), the plasmid is not so stable in the BL21 series of strains, and may be integrated into the genome, loss, mutation, etc., so construct a good vector It still needs to be stored in strains such as DH5α.

4. Optimization of culture conditions

Taking the E. coli expression system as an example, LB medium can be used for both resuscitation and seed culture. 

It is better to use a medium with higher amino acid content but lower sugar source during the fermentation stage, such as TB medium to promote protein Synthesis and accumulation. 

If an inducible promoter is used, an inducer such as IPTG should be added when the bacteria grow to the late logarithmic growth phase. 

After adding the inducer, the culture temperature needs to be lowered to 16-30°C. The lower the culture temperature, the slower the accumulation of protein and the less likely it is to produce inclusion bodies.

5. Purification column material selection

Each label has a corresponding purification column material. GST-tag uses glutathione gel, MBP-tag uses polysaccharide resin, CBD-tag uses chitin resin, and His-tag uses fillers coupled with divalent metal ions, the most common of which is coupled with nickel Ionic, commonly known as nickel column.

NEBExpress nickel column packing (S1428S) was newly launched in November 2019. There is also a pre-packed spin column form (S1427S/L), which is more suitable for small protein purification in the screening stage. Purification of 1mg His-labeled protein only takes 15 minutes.

Each 1 ml nickel column packing can purify ≥10 mg histidine-tagged protein;

Highly specific binding to proteins with His-tag, purity >95% after separation and purification;

The strong nickel ion binding makes it extremely resistant to EDTA and reducing agents, and is compatible with common detergent-based cell lysis reagents.