How to easily obtain a highly purified untagged recombinant protein

Isolation of highly purified untagged proteins is crucial in today’s R&D programs. Up to now, recombinant protein production approaches were often based on the use of “tags” to make protein purification (and solubility) more convenient. Unfortunately, these tags can be real experimental hurdles in downstream applications. Protein experts are now considering “tag-free” alternatives (including outsourcing) for producing untagged proteins more rapidly, more efficiently, and without jeopardizing the rest of the research project. Let’s see how this is possible.

Classical purification Strategy vs. IMPACT™ purification Strategy

Classical purification Strategy vs. IMPACT™ purification Strategy

As far as protein isolation is concerned, protein experts usually follow a classical 3-step approach:

  1. Affinity chromatography to obtain the recombinant protein
  2. A cleavage step with a protease to separate the untagged protein from his tag
  3. Another affinity chromatography in order to isolate the purified untagged protein from the tag and the protease

The main issues with such a classical system are the time required for it and the cost of expensive proteases. In addition, the tags used can prevent you from using your recombinant proteins in down-stream applications.

In order to bypass these hurdles and bring more reactivity in recombinant protein production stages, new protein expression systems based on the IMPACT technology* have recently been designed. This new strategy enables the expression of a target protein carrying a binding domain compatible with 1-step purification. Interestingly, this procedure does not require the use of proteases, thus reducing the overall costs of your protein production. (1)

Over the last decade, tebu-bio’s protein experts’ major aim has been to design time- and cost-effective protein expression and purification platforms. Our goal is to bring customers the latest technologies for high quality recombinant protein productions.

We recently upgraded our E. Coli protein expression with the IMPACT technology. Outsourcing your protein production to an innovative European platform such as tebu-bio is an efficient way to boost your protein production while better controlling human resources and expenses. It’s also a smart way to integrate the latest technologies without investing a cent in developments and long tests.

Illustration of the chemistry involved in purification using IMPACT method (inspired by http://www.ncbi.nlm.nih.gov/pubmed/26096506).

Illustration of the chemistry involved in purification using IMPACT™ method (inspired by ref. 1).

Let’s take a look at how we design the purification steps for your recombinant protein productions.

To summarize the strategy, your target protein is fused to a self-cleaving mutated intein sequence followed by a Chitin-Binding Domain (CBD). The CBD binds to an affinity column composed of Chitin resin with a high affinity and a high specificity. The addition of a thiol-containing compound such as DTT or ß-ME catalyzes the cleavage of the N-terminal extein sequence from the thioester intermediated formed with the intein sequence.

The result is the obtention of a completely untagged protein after hydrolysis. Occasional optimisation linked to the amino acids surrounding the intein sequence might be required. By interacting with tebu-bio’s lab scientists, who are highly familiar with this technology, you will be able to make the most of recent advances in protein production for your own proteins.

Looking for a highly untagged purified protein? Leave a message below or contact tebu-bio’s lab staff!

*IMPACT™ (Intein Mediated Purification with an Affinity Chitin-binding Tag) is a trademark of New England Biolabs.

Isabelle Topin, PhD
Written by Isabelle Topin, PhD
Isabelle Topin is a Project Manager at tebu-bio, who's expertise ranges from genes to proteins, through biochemistry and molecular, cellular, structural and animal biology.