Tehnical note compares methods used for fusion protein construction, tag cleavage involving the addition of protease, and subsequent steps involved for post-purification tag and protease removal
Bio-Rad Laboratories has announced the availability of a new technical note (tech note 5652A) entitled 'Profinity exact fusion-tag system performs on-column cleavage and yields pure native protein from lysate in less than an hour', which compares methods used for fusion protein construction, tag cleavage involving the addition of protease, and subsequent steps involved for post-purification tag and protease removal, to the novel Profinity Exact system.
Affinity tagging enables the expression and purification of large numbers of proteins using single-step purification methods.
However, all tags have the potential to interfere with the biological activity of a protein and to influence its behavior and need to be removed.
Using maltose-binding protein (MBP) as a model protein and the glutathione-S-transferase (GST) system for comparison, Bio-Rad has demonstrated in this technical note that its Profinity Exact fusion-tag system can rapidly produce pure, tag-free target proteins in a fraction of the time of standard methods currently available.
To demonstrate the advantages of the Profinity Exact system one-step protocol, Bio-Rad compared the purification process of MBP fused with either the GST-tag or the Profinity Exact tag.
To mimic the tag-removal capabilities of the Profinity Exact system, the GST-MBP fusions were also engineered with intervening thrombin or TEV cleavage sites.
Performance parameters tested in this study include the time required for obtaining tag-free MBP and final yield and purity of the purified protein.
Technical note 5652A is available from either a local Bio-Rad sales office or it may be downloaded from the Bio-Rad website.
