HD Transient Expression Service

A general capture and purification platform for tagless proteins based on a self-cleaving split-intein tag

Dr. David Wood is a Professor of Chemical and Biomolecular Engineering at Ohio State University. Dr. Wood is also a founder and co-owner of Protein Capture Science LLC, a company involved in the development and commercialization of self-cleaving tag technologies based on split inteins. In this webinar, Dr. Wood discusses the development of a single platform for purifying a broad range of protein types. Monoclonal antibody purification relies on a singular Protein A affinity platform, which is an efficient yet economical process. The applicability of this platform to antibody purification across the board is based on the ability of protein A to interact with a feature common to all antibodies, the Fc domain.

Currently, a similar platform is not available for the purification of all other non-antibody proteins; instead, their purification relies on the use of different columns based on the specific protein's features. The drawback of current protein purification platforms is the need for constant individualized optimization.

In conventional purification methods, proteins are labeled with affinity tags (e.g., His or Flag tag), which allow their purification by the use of standard solid matrix columns. The purified protein is additionally processed for tag removal by the use of endopeptidases. Removal of tags from proteins intended for therapeutic use is a requirement, given their potential for eliciting adverse immune responses. Some of the disadvantages of this approach relate to this last step, as endopeptidases may be expensive, require additional processing, could lead to protein loss, or may not work efficiently.

Therefore, Dr. Wood's work aimed at developing a platform able to purify a broad range of proteins by using a single column. As a solution to the inherent problems associated with the use of proteases, Dr. Wood envisioned a process where the tag would cleave itself off from the protein of interest. This new purification workflow is enabled by the use of an intein based universal affinity capture process and allows the recovery of tagless proteins.

What are Inteins?

Inteins were first discovered in the 1990s. Inteins are intervening protein sequences that catalyze their own splicing leading to the final functional protein product. The mechanism for inteins’ splicing was rapidly resolved, and it was determined that the process was inducible. For example, cleavage of inteins from a protein's N-terminus is induced by thiol, while C-terminal cleavage is induced by changes in temperature or pH.

Therefore, Dr. Wood envisioned a new workflow, where inteins incorporated between an affinity tag and a protein of interest would enable removal of tags without the need for extraneous protease activity. In this system, following protein purification, a washing step under specific pH and temperature conditions would be the only requirement for tag elimination.

Developing an Intein Based Tagless Protein Purification System

Through various genetic engineering strategies, Dr. Wood was able to develop a mini-intein with accelerated cleavage rate, which was sensitive to both pH and temperature changes. One advantage of this approach is that it enables the purification of other proteins that may be engaging in non-covalent interactions with the tagged-intein-recombinant protein of interest (e.g., subunits within an RNA polymerase complex).

Dr. Wood was able to demonstrate the efficiency of this approach for purifying different recombinant proteins expressed in bacterial cells. However, the use of inteins for expression and purification of proteins from mammalian cells presented challenges related to the unwanted and premature cleavage induced by growth conditions, which significantly reduced protein yield.