The PURE system is more robust and convenient than most extract-based systems for many
in vitro applications. The immediate advantage is the significantly reduced level of all contaminating activities. It can be used to express a wide range of protein targets and has the capacity for a yield of more than 100 μg/ml. The activity of the synthesized protein can often be directly assayed without purification due to the low background activity of the translation mixture. All recombinant protein factors inside the PURE system are His-tagged, in some cases allowing the synthesized protein to be “reverse-purified” (Figure 1) (1). The purity of this system allows it to withstand more than five freeze-thaw cycles without losing its efficiency, further extending its shelf life (Figure 2) (Cantor, E., unpublished observation).
Figure 1: Expression and reverse purification of DHFR (A) and T4 DNA Ligase ( NEB #M0202) (B) using PURExpress® ( NEB #E6800). 125 µl reactions were carried out according to recommendations in accompanying manual. Samples were analyzed on a 10–20% Tris-glycine gel and stained with Coomassie Blue. Note that in both cases, the desired protein can be visualized in the total protein fraction. The red dot indicates the protein of interest. Marker M is the Protein Ladder ( NEB #P7703)
Figure 2: PURExpress retains activity after multiple freeze-thaw cycles. Solutions A and B were subjected to four rounds of freeze-thaw treatment and then used to set up standard reactions according to manual recommendations. The red dot indicates the protein of interest. Marker M is the Protein Marker, Broad Range ( NEB #P7702) References
Shimizu, Y., et al. (2001) Cell-free translation reconstituted with purified components.
Nat. Biotechnol. 19, 751–755. PMID: 11479568
PURExpress® is a registered trademark of New England Biolabs, Inc.