Here we describe a strategy for isolation of multiprotein complexes from human HeLa S3 cells in a scale and purity optimized for characterization by mass spectrometry. For this purpose, we use stably expressed One-STrEP-tag® fusion proteins. This approach was successfully used in characterization of histone chaperone Asf1 complexes (Groth et al., 2007), and we have recently optimized it further. Using this protocol we routinely obtain complexes in amounts sufficient for visualising single protein bands by Coomassie Blue staining.
The Strep-tag®II (SAWRHPQFGG) and its “double” sister, the One-STrEP-tag (tandem arrangement of Strep-tag®II, here called OneStrep), are reasonably small protein tags that usually do not influence protein folding and function. However, always check by appropriate control experiments that the tagged protein is functional in vivo. Vectors for both N- and C-terminal fusion are available from IBA Tagnology (Germany), who has developed these tags and binding matrices. We recommend using the OneStrep-tag for purification of protein complexes from mammalian cells. The Strep-tag® has a strong affinity towards engineered streptavidin (Strep-Tactin®) which allows efficient one-step purification with high stringency washing to obtain highly pure protein complexes. Elution of the complexes by competition with D-biotin is efficient and can be done in a variety of buffer conditions allowing biochemical active complexes to be preserved. For more theoretical background and a protocol for purification of recombinant Strep-tag® fusion proteins from E.coli please refer to Schmidt and Skerra (2007).
In comparison to other commonly used purification strategies (i.e. HA and FLAG double-tag purification, Tagami et al., 2004), the advantages of the OneStrep-tag are:
- Purification in only one step;
- Strong affinity to Strep-Tactin® matrix allowing stringent washing;
- Highly efficient elution;
- Broad range of available Strep-Tactin® matrices including columns for gravity flow and HPLC as well as magnetic beads and spin-columns for small-scale purification;
- And cost-effectiveness.
Note that a two-step purification strategy may be advantageous to identify weak interactors.
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