Bio-Rad Laboratories has published methods for using the Profinity Exact fusion-tag system to express recombinant proteins in eukaryotic and bacterial cells.
The methods also show how the Profinity Exact system can be used to obtain tag-free purified recombinant proteins in a process that takes less than an hour.
Several bulletins that describe the methods are available for download at the company's website by entering bulletin number 5656, 5652, 5813, 5770 or 5928 into the search box.
The Profinity Exact fusion-tag system employs a novel combination of the Profinity Exact tag and a tag-binding protease immobilised on the purification resin.
The 8 kD Profinity Exact tag is fused to the N-terminus of the protein of interest and is readily removed by a single on-column cleavage step activated on the immobilised protease by fluoride or azide ions in the elution buffer.
The tag remains bound to the protease while the tag-free protein is eluted.
The single-step tag removal and purification process is said to be unique to the Profinity Exact system: the immobilised protease binds and cleaves the tagged protein, allowing tag-free protein to be recovered from a single column pass-through.
The Profinity Exact fusion-tag system has been optimised extensively in E coli to demonstrate the speed of purification and the complete removal of the fusion tag from the protein of interest.
Bulletin 5656 describes how the Profinity Exact fusion-tag system can be used in E Coli.
Bulletin 5652 demonstrates the effectiveness of cleavage for a recombinant protein isolated from E Coli and its greater efficiency compared with the gluthathione-S-transferase (GST) tag purification system.
The Profinity Exact fusion tag is available from Bio-Rad in an E coli expression vector.
Bulletin 5813 details how the Profinity Exact fusion tag can be incorporated into vectors that use alternate promoters, drug selection markers, or express in different hosts.
The method employs Stratagene's QuickchangeXL site-directed mutagenesis kit to incorporate the tag into the Gateway pDest17 E Coli expression vector from Invitrogen.
Bulletin 5813 provides a simple protocol that researchers can use to tailor the Profinity Exact fusion-tag system to meet their individual research requirements.
In bulletin 5770 Bio-Rad scientists make use of the Profinity Exact fusion-tag system to generate an N-terminal tagged cystathionine beta-lyase (MetC) from the bacterium Shewanella oneidensis.
The protein is subsequently purified in milligram quantities, free of tag using the Bio-Rad Profinia protein purification system, an automated affinity chromatography technology that complements the Profinity Exact fusion-tag system.
With a 10ml sample of whole cell lysate applied for purification, the Profinity Exact tag is efficiently cleaved in 12min at room temperature at the elution step, while typical fusion tag removal processes often takes hours post-affinity purification.
The purified tag-free MetC is also functionally active in vitro.
Bulletin 5928 details how Profinity Exact fusion-tagged proteins can also be readily expressed in both insect and HeLa cells and the fusion tag can easily be removed with a single cleavage step.
In addition, the functionality of the protein expressed in HeLa calls is not compromised by the addition and subsequent removal of the fusion tag.
The application notes described demonstrate the value of the Profinity Exact and Profinia systems for researchers looking for easy solutions to affinity tagging purification of proteins from prokaryotic and eukaryotic sources.
