The flexibility in adding extra amino acids upstream of Cytiva™ Protein Select™ tag can facilitate detection, purification, analysis, or solubility.

Cytiva™ Protein Select™ tag is a 36-amino acid self-cleaving tag that enables the affinity chromatography  purification of recombinant proteins using Cytiva™ Protein Select™ resin.

During an affinity step performed with Cytiva™ Protein Select™ resin, the protein self-cleaves from the tag and elutes with no residual tag amino acids. The cleavage site is located between the last amino acid of the tag and the first amino acid of the target protein (Fig 1).

Extra amino acids such as signal peptides or additional tags can be placed upstream of the Cytiva™ Protein Select™ tag to facilitate your work during and after protein expression (Fig 1).  After the purification with Cytiva™ Protein Select™ resin, you will still benefit from the Cytiva™ Protein Select™ technology and obtain a native protein with no remaining tag amino acids.

Here we discuss how to design the construct with extra amino acids to:

  • Control protein secretion and translocation.
  • Improve purity and/or to concentrate your sample.
  • Facilitate protein detection or analysis.
  • Increase stability towards proteolytic activity.
  • Increase protein solubility.

Adding a signal peptide for controlling protein secretion and translocation

Signal peptides can be placed upstream of the Cytiva™ Protein Select™ tag to target and direct newly synthesized proteins to specific locations within the cell or for secretion outside the cell (Fig 1).

In principle, you can use any signal peptide with the Cytiva™ Protein Select™ tag—we have not seen any signal peptide constraint or limitation. In our tests, IgG kappa, CD 36, and interleukin-2 signal peptide sequences worked well.

Since all amino acids upstream of the Cytiva™ Protein Select™ tag are removed after purification with Cytiva™ Protein Select™ resin, you also remove the risk of residual signal peptide due to miscleavage of signal peptide.

It is also a good idea to have a short amino acid linker between the signal peptide and Cytiva™ Protein Select™ tag, to minimize the risk of truncation of the tag caused by inexact cleavage of the signal peptide.

 

Fig 1. An additional signal peptide can be added upstream of to the Cytiva™ Protein Select™ tag.

 

Adding a tag to improve purity and/or to concentrate your sample

When one affinity purification with Cytiva™ Protein Select™ technology does not achieve the desired purity, or if the sample volume is too large for being applied to Cytiva™ Protein Select™ resin (as short sample loading time is important with this resin), consider adding a histidine tag (his-tag) and performing an immobilized-metal affinity chromatography (IMAC) before the purification using Cytiva™ Protein Select™ resin.

The IMAC step serves both as a purification and a concentration step. After the purification step with Cytiva™ Protein Select™ resin, you will obtain a native pure protein with no trace of the tag amino acid.

Figure 2 shows how interleukin 1β (IL-1β) is constructed with both his-tag and Cytiva™ Protein Select™ tags to enable the above-described protocol. A two-step purification is performed, first using an IMAC resin followed by the Cytiva™ Protein Select™ resin.

 

Fig 2. Protein constructed with a 6 × his-tag and Cytiva™ Protein Select™ tag.

 

The example below shows the purification of IL-1β with a dual tag (Fig 2) using an IMAC resin as the first step with a 12 mL sample load. We further purified the eluted protein from IMAC purification and cleaved the tags on a HiTrap™ Protein Select™ 1 mL column with a 3 mL sample load (Fig 3).

Figure 4 shows the SDS-PAGE analysis after each purification step. You can see that the purity of the protein is significantly higher after the two-step purification (lane 6 in Fig 4) than the eluted protein after IMAC (lane 3 in Fig 4) . We loaded a reduced sample volume (from 12 mL to 3 mL) onto HiTrap™ Protein Select™ column since a concentrated sample is obtained after the IMAC step. Because Cytiva™ Protein Select™ resin is compatible with imidazole, the eluted protein from IMAC purification can be directly loaded onto HiTrap™ Protein Select™ column with no need for buffer exchange.

After the two-step purification, we obtained 6.5 mg of native protein, free from tag amino acid traces, with a purity of 96%.

 

Fig 3. Two-step protein purification using an (A) IMAC step followed by (B) a step using Cytiva™ Protein Select™ resin.

 

Fig 4. SDS-PAGE analysis of wash and eluate samples for both purification steps.

 

Adding a tag to facilitate detection or analysis

You can place a small tag, such as the his-tag or FLAG-tag upstream of the Cytiva™ Protein Select™ tag and these tags can be used for detection or analysis. For example, you can incorporate a his-tag in your protein construct and use an anti-his antibody to detect your tagged target protein in cell lysate using, for example, Western blotting or using surface plasmon resonance (SPR) technology.

In the following example we added a histidine epitope tag upstream of the Cytiva™ Protein Select™ tag to facilitate analysis of the amount of full-length recombinant protein in crude cell extracts. Since crude cell extracts are complex samples with large amounts of different protein molecules, you need a binder with high specificity (such as an antibody) to detect or quantitate the target protein.

Using anti-his antibody on a Biacore™ (SPR) instrument, you can quantitate the target protein containing both his-tag and Cytiva™ Protein Select™ tags, by measuring the SPR signal (Fig 5) in a complex solution.

Both his-tag and Cytiva™ Protein Select™ tag are removed during the purification on Cytiva™ Protein Select™ resin, resulting in a native protein without residual tag amino acids.

 

Fig 5. (A) Protein construct with upstream (his)6-tag allowing Biacore™ SPR analysis using anti-his antibodies. (B) Calibration curve using purified fusion protein as standard to quantitate his-tag target protein in a complex solution.

 

Adding amino acids to further increase stability of Cytiva™ Protein Select™ tag to proteolytic activity

An intact Cytiva™ Protein Select™ tag is required for ensuring the expressed tagged protein to bind to the Cytiva™ Protein Select™ resin. It is therefore important that tagged proteins in cell culture media are stable against truncation of the tag from, for example, proteases present in culture media, before the purification step using Cytiva™ Protein Select™ resin.

The Cytiva™ Protein Select™ tag is in general very stable, but in cases where truncation occurs in, for example, cell culture or during sample storage, extra amino acids upstream of Cytiva™ Protein Select™ tag can potentially reduce the risk of truncation. The extra amino acid can be other tags such as a 6 × his-tag or flexible linkers with, for example, repeats of (Gly-Gly-Ser-Gly) sequence (Fig 6).

 

Fig 6. Examples of adding amino acids to reduce the truncation of Cytiva™ Protein Select™ tag. (A) 6 × his. (B) A flexible peptide linker.

 

Adding a tag to increase the solubility of the tagged protein

When working with recombinant proteins that are prone to forming insoluble inclusion bodies in E. coli expression systems, you can add solubility tags to help solubilize recombinant proteins during protein production and reduce aggregation. Using Cytiva™ Protein Select™ technology, you can also add a solubility tag upstream of the Cytiva™ Protein Select™ tag, which will not only enhance the solubility but also deliver a native protein without any tag residuals within one step of purification. The cleavage rate might be slightly reduced.  However, with improved solubility of target protein, the overall yield might still be improved.

You might consider adding a linker (several amino acids) between the two tags to reduce the risk of steric hindrance, for example, repeats of Gly-Gly-Ser-Gly sequence (Fig 7).

 

Fig 7. Example of a construct that facilitates the solubility of the tagged protein.

 

Summary

The article discusses the advantages of adding extra amino acids upstream of the Cytiva™ Protein Select™ tag. The increased flexibility can aid in protein detection, purification, analysis, and increasing solubility.

Key points include:

  1. Control of protein secretion and translocation: Signal peptides can be added upstream to direct proteins to specific cell locations or secretion pathways.
  2. Improved purity and concentration: Adding a his-tag allows for an initial immobilized metal affinity chromatography (IMAC) step, increasing purity and concentration before using the Cytiva™ Protein Select™ resin for final purification.
  3. Facilitated detection or analysis: Tags like the his-tag or FLAG-tag can be added for easier detection and analysis, such as using anti-his antibodies in Western blotting or SPR techniques.
  4. Increased stability to proteolytic activity:  Extra amino acids can protect the Cytiva™ Protein Select™ tag from truncation by proteases during cell extraction and storage.
  5. Increased protein solubility: Soluble tags can be added to prevent aggregation and improve solubility in recombinant protein production, especially in systems like E. coli.

 

We show detailed examples and experimental results, illustrating the benefits of these modifications in achieving high purity and yield of native proteins free from tag residues.

Related resources

Memo 31246362

CY48092