General Information of Different Tags


View:581 Time:2018-06-05


Affinity tag fusion expression provides a very simple and convenient purification tool for recombinant protein purification. It has the advantages of high binding specificity, mild elution conditions, high versatility, and high purification yield and purity. Its role is mainly the following 6 points.

1. Increase Yield of Recombinant Proteins

  • Difficult-to-express proteins can be successfully expressed. For example, one protein can not be expressed with no-tag vector, but can be successfully expressed with a tagged vector. We could try multiple tags at the same time instead of tying one by one, to obviously improve protein expression success and efficiency.
  • Expression yield from low to high. A difference of 10 to 100 times of expression efficiency is possible. Such common tags include GST-tag, MBP-tag, SUMO-tag and so on.

2. Improve Protein Stability

Certain amino acid sequences at N-terminus or C-terminus of some target proteins will affect the degradation of the proteins themselves. Some tags are resistant to the degradation of proteases in the host, so that the fusion proteins will be more resistant to protease degradation in the host, thereby increasing stability. Such common tags include GST-tag, MBP-tag, SUMO-tag, B2M-tag and so on.

3. Enhance Protein Solubility

E. Coli expression has a high probability of forming inclusion bodies. Fusion tag is a good way to improve solubility, especially when conventional optimization parameters do not work. The conventional optimization parameters include: exchange of expression host, decrease of induction temperature, adjustment of inducer dosage, and expression of chaperones. Such common tags include GST-tag, MBP-tag, SUMO-tag, NusA-tag and so on.

4. Promote Correct Protein Folding

Protein folding is directly related to the protein's spatial structure and biological activity. Generally, whether a protein can be correctly folded depends on its own amino acid sequence. However, these affinity tags can be used as an adjunct to increase the probability of correct protein folding. This is consistent with improved solubility. If the folding is incorrect, it can easily form inclusion bodies. Such common tags include GST-tag, MBP-tag, SUMO-tag, NusA-tag, Trx-tag and so on.

5. Enhance Protein Immune Activity

In general, some tags themselves contain multiple epitopes. Especially for small proteins, such as proteins below 10kDa, it is difficult to produce antibodies by immune alone, and fusion of a large tag can significantly improve immunogenicity. Such common tags include GST-tag, MBP-tag, NusA-tag.

6. Protein Follow-up Applications

Fusion tags make some experiments more convenient and concise, such as WB, GST/His pull down, IP/Co-IP, functional ELISA detection and so on.

Features of Several Common Tags

1. His-tag

It is currently the most popular affinity tag for high-throughput protein purification, and its advantages mainly include 4 points.

  • Hardly affect the physicochemical properties of the target protein.
  • The tag is very small and will not change the solubility of the target protein.
  • It has little effect on the protein structure after the target protein is crystallized.
  • With IMAC purification, the operation is very simple and versatil.

2. FLAG-tag

In addition to His-tag, it is another widely used short peptide tag, and its advantages mainly include the following 3 points.

  • The molecular weight is very small, so it will not cover the target protein and will not change the structure and function of the target protein.
  • It is naturally hydrophilic and easily locates the surface of the fusion protein for easy detection of corresponding antibody.
  • The tag itself contains an enterokinase (EK) cleavage site (DDDDK) that can cleave the tag using enterokinase, and end cutting can result in no extra amino acid residues.

3. GST-tag

It is currently the most common type of large tag, and its advantages include the following 5 points.

  • Improve protein yield and stability.
  • Enhance protein solubility.
  • Promote proper protein folding.
  • Increase protein immunogenicity.
  • Purified under mild conditions (non-denatured).

4. MBP

In addition to GST-tag, it is another commonly used large tag, and its advantages mainly include the following 5 points.

  • Improve protein yield and stability.
  • Enhance protein solubility.
  • Promote proper protein folding.
  • Increase protein immunogenicity.
  • Purified under mild conditions (non-denatured).

5. Combination Tags

Such as His-GST-tag, His-SUMO-tag, His-MBP-tag, these tags have specific dual-tag features: the His-tag is conducive to purification (denatured and non-denatured), and the large tag (GST, SUMO, MBP) promotes proper protein folding, optimize protein yield and stability.

 

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