While affinity tags (e.g., His-tag, GST-tag) are commonly used to facilitate protein purification, there are scenarios where tag-free proteins are desired, especially for structural studies, therapeutic applications, or functional assays where the tag might interfere with the protein's activity. Here are several strategies to obtain tag-free proteins:

1.  Protease Cleavage Sites:

          ● Design: Incorporate a specific protease cleavage site (e.g., TEV protease, thrombin, or Factor Xa) between the purification tag and the target protein.

          ● Expression and Purification: Express the fusion protein with the tag, purify it using affinity chromatography, and then treat the purified protein with the specific protease to cleave off the tag.

          ● Secondary Purification: Perform a secondary purification step (e.g., size exclusion chromatography or ion exchange chromatography) to separate the cleaved tag from the target protein.

 

2.  Self-Cleaving Tags:

          ● Example: The intein system, where the tag is fused to an intein that undergoes self cleavage under specific conditions (e.g., changes in pH or temperature).

          ● Process: Express the fusion protein, purify it, and then induce self-cleavage to release the tag-free target protein.

 

3.  Native Protein Expression:

          ● No Tag Addition: Express the target protein without any fusion tag using a native expression system.

          ● Challenges: This method may be challenging due to lower expression levels and difficulty in  purification. It often requires optimizing other purification methods, such as ion exchange, hydrophobic interaction, and size exclusion chromatography.

 

4.  Use of Signal Peptides for Secreted Proteins:

          ● Strategy: In systems where the target protein is secreted (e.g., yeast, mammalian cells), the native signal peptide can direct the secretion of the protein into the culture medium.

          ● Purification: Collect the culture medium and purify the secreted protein using conventional chromatography techniques, without the need for a purification tag.

 

5.  Affinity Tag Removal Systems:

          ● Commercial Kits: Some companies offer kits that provide proteases and specific cleavage conditions tailored for efficient tag removal and subsequent purification.

          ● Examples: Kits from companies like Sigma-Aldrich, Novagen, and Qiagen.

Steps for Obtaining Tag-Free Proteins Using Protease Cleavage Sites

1.  Construct the Expression Vector:

          ● Insert the target gene into an expression vector with an affinity tag (e.g., His-tag) and a protease cleavage site (e.g., TEV, thrombin, or Factor Xa) upstream of the target protein.

 

2.  Express the Fusion Protein:

          ● Transform the expression vector into the appropriate host cells (e.g., E. coli, yeast, mammalian cells) and induce protein expression.

 

3.  Purify the Fusion Protein:

          ● Use affinity chromatography to purify the tagged fusion protein from the cell lysate or culture medium.

 

4.  Cleavage of the Tag:

          ● Treat the purified fusion protein with the specific protease to cleave off the affinity tag. Optimize conditions (e.g., temperature, pH, incubation time) to achieve efficient cleavage.

 

5.  Secondary Purification:

          ● Separate the cleaved tag and the protease from the target protein using techniques such as size exclusion chromatography, ion exchange chromatography, or another round of affinity chromatography targeting the tag or the protease.

 

6.  Quality Control:

          ● Verify the purity and integrity of the tag-free protein using SDS-PAGE, Western blotting, and mass spectrometry. Assess the protein's activity and function through relevant assays.

Obtaining tag-free proteins involves strategic planning during the design of the expression construct, incorporating a protease cleavage site, and optimizing purification protocols. Methods like protease cleavage, self-cleaving tags, native expression, and the use of signal peptides enable the production of high-quality, tag-free proteins suitable for various applications.

GenCefe provide one-stop solutions from codon optimization, gene synthesis, to protein expression and purification. In addition to four conventional expression systems of bacterial, yeast, insect cell, and mammalian cell, we have also developed a patented cell free expression technology, which is suitable for the production of difficult proteins such as toxic proteins and membrane proteins.