Tag-free Protein Production

Compared with tagged proteins, tag-free proteins have identical sequences and similar crystal structures to native proteins. Recombinant proteins with exogenous amino acids may cause immunogenicity if they are used in animal cells, and they have limited chance to pass the FDA approval. In addition, the result of functional domain and crystal structure analysis can be affected by large tags, which can limit the folding of recombinant proteins and significantly impact the protein structure and biological functions.

The tag-free protein service offered by Biologics International Corp (BIC) provides untagged proteins more rapidly and more efficiently. The main advantages of our service is the assurance that there are no exogenous amino acids or proteases in our products and the elution fraction has high homogeneity. Once we are provided with the DNA sequences, our dedicated teams strive to work with each customer to successfully develop the best possible tag-free protein. For any question regarding our tag-free protein service, please contact us. We will be happy to help you.

Tag-free Protein Purification

Our tag-free protein purification service starts with the synthesis of genes without tags, followed by ion exchange, which is usually the first step in the purification process. After selecting the proper initial conditions (e.g., pH and ionic strength), elution conditions (e.g., gradient volume, concentration and slope) and matrix, we can purify large amounts of the target protein. Depending on the properties of the target protein, subsequent steps will invovle several of the following methods until we meet your specific needs.

  • Ammonium sulfate precipitation
  • The solubility of proteins changes at different salt concentrations. With the increase of salt ion concentration, the solubility of the proteins first increases and then the proteins begin to precipitate. These two effects are termed "salting in" and "salting out", respectively. Due to ammonium sulfate’s high solubility, ability to stabilize protein structures, relatively low density, and inexpensive cost, it is especially useful as a precipitant.

  • Size-exclusion chromatography
  • Size-exclusion chromatography (SEC), also known as molecular sieve chromatography, is widely applied in separating large molecules or macromolecular complexes (such as proteins). The SEC method separates different molecules by size and weight, and it has several advantages, such as its good separation of large molecules from the small molecules, its short and well-defined separation times, and the fact that various solutions can be applied without interfering with the filtration process.

  • Hydrophobic interaction chromatography
  • Hydrophobic interaction chromatography (HIC) separates proteins using the properties of hydrophobicity. When proteins pass through the HIC column, hydrophobic groups (such as phenyl, octyl, or butyl) attached to the stationary column are able to interact with and bind to these proteins. The principle of protein adsorption onto HIC media is complementary to that of ion exchange.

And More

We are able to produce and purify various kinds of protein, including recombinant phosphoproteins, glycoproteins and recombinant antibodies, which require the use of several other methods. If you have any questions or concerns, please speak with our scientists, who are experts in the use of these technologies, and they can help customize a protocol that is the most suitable for your specific proteins.

Case Study

At BIC, we have established a fast and flexible purification protocol for producing tag-free proteins without jeopardizing the rest of the research project. The following case study illustrates the process of a tag-free project for protein S. After six rounds of purification, our professional team had purified the protein S at a the concentration as high as 12 mg/ml and a purity greater than over 95%, which met the customer’s requirement.

Tag-free protein production

Fig. 1 SDS-PAGE analysis of protein expression

Lane M: Protein marker; Lane 1: Contrast; Lane 2: Induction at 15 ℃ overnight; Lane 3: Induction at 28 ℃ overnight; Lane 4: Induction at 37 ℃ for 4 h

Tag-free protein purification

Fig. 2 SDS-PAGE for the purification of the collected Flow-through after concentration

Lane M: Protein marker; Lane 1-16: Elution and collection via size-exclusion chromatography

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