(Twin-) Strep-tag®/Strep-Tactin® affinity purification kit BioVector NTCC质粒载体菌种细胞基因保藏中心

(Twin-) Strep-tag®/Strep-Tactin® affinity purification kit BioVector NTCC质粒载体菌种细胞基因保藏中心 Purification of (Twin-) Strep-tag® fusion proteins with Strep-Tactin® matrices

(Twin-) Strep-tag®/Strep-Tactin® affinity purification

The Strep-tag® purification system is based on the highly selective binding of engineered streptavidin,
called Strep-Tactin®, to Strep-tag® II fusion proteins. This technology allows one-step purification of
almost any recombinant protein under physiological conditions, thus preserving its biological activity. The
Strep-tag® system can be used to purify functional Strep-tag® II proteins from any expression system
including baculovirus, mammalian cells, yeast, and bacteria. Unique Strep-Tactin® affinity columns have
been developed for this purpose and the corresponding operating protocol is described below. Streptag®/Strep-Tactin®
affinity purification should not be performed discontinuously in batch mode which
would result in significantly reduced protein purity and yield in comparison to column chromatography.
Further, prolonged batch incubations increase the risk of proteolytic degradation of the target protein
including cleavage of the tag. Because of its small size, Strep-tag® generally does not interfere with the
biological activity of the fusion partner. Thus, removal of the tag becomes superfluous.
The Twin-Strep-tag® is a dimeric version of the Strep-tag®II and therefore binds with the same selectivity
to Strep-Tactin® but with a higher affinity. This higher affinity allows the purification of Twin-Strep-tagged
proteins even from batch or cell culture supernatants with good yields. In addition the Twin-Strep-tag®
tolerates higher amounts of detergents and salts in buffers compared to Strep-tag®II. Since the overall
conditions for Strep-tag®II and Twin-Strep-tag® are the same, the following protocol can be used for both
tags.
Short Protocol of the Strep-Tactin® chromatography cycle
Perform all operations at a temperature amenable to the stability of your recombinant protein (between 4
°C and 30 °C). To achieve optimal purification results, comply with the specified volumes and their ratios
(column bed, washing volumes etc., see page 3). At low expression levels, increase applied cell extract
volumes to take advantage of the column capacity, without changing other volumes.


Table 1. Recommended buffer volumes for chromatography on Strep-Tactin® columns
*Adjust protein extract volume according to binding capacity of the column (please refer to the appropriate data
sheet) and apply the extract as concentrated as possible in the recommended volume range. Note that these
volumes are average values which can be different for certain proteins.

Biotin in cell culture media
Please note that biotin binds with high affinity to Strep-Tactin® thereby efficiently competing binding of
(Twin-) Strep-tag®II. This binding is in addition irreversible and does to not allow regeneration of the StrepTactin®
column (in contrast to bound desthiobiotin).
Especially media for mammalian cell or insect cell cultivation may contain significant amounts of biotin.
Thus, if proteins are secreted to the culture medium, biotin must be masked by the addition of avidin (or
biotin should be removed by dialysis or gel filtration) prior to Strep-Tactin® chromatography.

[Supplier来源] http://www.biovector.net