Unlock the potential of high-affinity purification with Strep-Tactin®XT

Strep-Tactin® and its high-affinity counterpart, Strep-Tactin®XT, are streptavidin mutants, which bind strep-tagged proteins reversibly. The differences lie in the binding strength of the possible tag-ligand combinations.

Strep-Tactin®XT stands out as the high-affinity option tailored for purifying strep-tagged fusion proteins. With binding affinities reaching the picomolar range for Twin-Strep-tag®, it retains reversible binding properties and ensures gentle recovery of immobilized proteins. This versatility extends to various protein classes and sizes, enabling efficient purification of challenging and low-abundance proteins alike. Strep-Tactin®XT fused to magnetic beads, MagStrep® Strep-Tactin®XT beads, is the ideal tool for fast small-scale batch purification in reaction tubes or 96-well plates, and protein complex identification via pull-downs.

In particular, Strep-Tactin®XT fused to 4Flow® agarose, Strep-Tactin®XT 4Flow® high capacity resin, offers exceptional efficacy in isolating strep-tagged proteins from diluted cell extracts, enabling thorough washes with substantial volumes of buffer. The binding affinity is stable across a broad pH spectrum and compatible with diverse buffer conditions, ensuring versatility. Moreover, the high stability of the resin ensures effective binding over long periods of time.

Higher protein yields with Strep-Tactin®XT

Differently sized proteins tagged with Strep-tag®II or Twin-Strep-tag® were purified with Strep-Tactin® and Strep-Tactin®XT. In all examples, Strep-Tactin®XT showed the highest binding capacity (1.6-2 fold higher compared to Strep-Tactin®; left graph). In another experiment, a Fab fragment fused to either Twin-Strep-tag® or Strep-tag®II was added to cell lysates at a concentration of 0.6 mg/ml. The amount of purified protein was more than 3-fold lower for the Strep-tag®II-fused Fab fragment if Strep-Tactin® was used (right graph). This could partially be rescued with a Twin-Strep-tag®, which has a higher affinity to both resins. Nevertheless, using Strep-Tactin®XT high capacity resulted in the highest yield independent from affinity tag.

The maximum binding capacities of Strep-Tactin® or Strep-Tactin®XT high capacity was determined with gravity flow columns for three differently sized proteins fused to either Strep-tag®II or Twin-Strep-tag®.

A Fab fragment fused to either Strep-tag®II or Twin-Strep-tag® was added to cell lysates at a concentration of 0.6 mg/ml and subsequently purified using Strep-Tactin® or Strep-Tactin®XT high capacity FPLC columns.

Cost-effective protein purification

Strep-Tactin®XT resins are the most cost-effective resins due to their higher binding capacity. As compared to Strep-Tactin® resins, Strep-Tactin®XT resin yields more than 2-fold more protein per ml resin and costs up to 54% less per milligram of purified protein. Furthermore, Strep-Tactin®XT prevents the unwanted leakage of the target protein during the washing steps.

Most efficient binding: Strep-Tactin®XT:Twin-Strep-tag®

Strep-Tactin® and Strep-Tactin®XT were loaded with twin-strep-tagged red fluorescent protein mCherry. Strep-Tactin®XT binds the protein directly in the upper part (right column), while Strep-Tactin® resin binds over nearly the entire resin bed (left column). The lower affinity of the tag to Strep-Tactin® causes the protein to detach more easily, resulting in a lower binding capacity as the protein flows out of the column.

During the washing steps, the protein is tightly bound by Strep-Tactin®XT and no protein is lost during purification. In contrast, significant mCherry loss is observed with Strep-Tactin® resin. This result highlights that Strep-Tactin® resins are not compatible with large samples or washing volumes. In the purification steps, due to the lower affinity, the protein detaches and runs off the column as the column volume is increased.

High stability: Strep-Tactin®XT outperforms Strep-Tactin®

A significant contrast between Strep-Tactin® and Strep-Tactin®XT resins lies in their regeneration durability. Following just a few regeneration cycles with 0,1 M NaOH, Strep-Tactin® binding decreases, resulting in lower protein yields. In contrast, the high stability of the Strep-Tactin®XT 4Flow® high capacity allow long-term use without performance loss.

Long-term use of Strep-Tactin®XT is possible due to its high stability

High affinity of Strep-Tactin®XT enables SPR analysis and BLI

Strep-Tactin®XT can be efficiently immobilized on SPR and BLI sensors and enables capturing of TST-fused ligands with exceptionally high affinity in pM range. The system allows kinetic analyses of strong binding analytes with long dissociation times and thus overcomes the current limitations of other affinity tag-based capture systems, such as the His-tag. The possibility to capture diverse ligands even directly from culture media and a simple regeneration procedure of the biosensors add major value to the application of Strep-Tactin®XT in optical biosensor assays.

Read more about Strep-Tactin®XT technology in downstream applications in this application note. Also, find out why Twin-Strep-Tag® in combination with Strep-Tactin®XT is a better alternative to the commonly used Avi-tag in SPR here.

SPR chips were coated with Strep-Tactin® or Strep-Tactin®XT and used for immobilizing a CD45 nanobody tagged with Twin-Strep-tag®. The results show that immediately after capture, the nanobody dissociates from Strep-Tactin®. In contrast, with Strep-Tactin®XT the nanobody is bound stably over a long-period of time. These data demonstrate that Strep-Tactin®XT can be used to analyze high-affinity interactions with slow dissociation rates.

Convenient assays and high-throughput screenings with Strep-Tactin®XT

Strep-Tactin®XT coated microplates ensure convenient assays and high-throughput screenings for strep-tagged biomolecules. Especially, the combination Twin-Strep-tag®:Strep-Tactin®XT is highly stable with a T_1/2 of 13 hours and affinity in low pM range. The tag-ligand combination is an efficient and elegant option for an antibody-free immobilization of proteins.

Moreover, the immobilized biomolecules are presented to interaction partners in a uniform manner, which results in reliable and highly reproducible assay formats. This minimizes non-specific binding concomitant with minimal coefficients of variation. Hence, the Strep-Tactin®XT coated microplates are a precise but cost-effective tool for high-throughput screenings and diagnostic assays.

Efficient immobilization of (Twin-)Strep-tag®II fusion proteins on Strep-Tactin®XT
(A) Schematic of the oriented binding of recombinant proteins with N-terminal or C-terminal Strep-tag® during an fluorometric microtiter plate (MTP) assay. (B) Using a fluorometric MTP assay the maximum binding capacity of Strep-Tactin®XT was examined and compared with Strep-Tactin®. Bacterial alkaline phosphatase (BAP, BAP-Strep-tag®II and BAP-Twin-Strep-tag®) was applied onto microplates each coated either with Strep-Tactin® or Strep-Tactin®XT. After washing, the amounts of bound BAP were determined demonstrating that Strep-Tactin®XT recovers significantly more protein. (C) Comparison of the relative maximal binding capacity showing the efficient immobilization of proteins on Strep-Tactin®XT.

References for Strep-Tactin®XT

Protein purification

The adaptive landscape of a metallo-enzyme is shaped by environment-dependent epistasis (Anderson, et al. (2021). Nature)
CryoEM of RUVBL1–RUVBL2–ZNHIT2, a complex that interacts with pre-mRNA-processing-splicing factor 8 (Serna, et. al.(2022). Nucleic Acids Research)

High-throughput purification

Affinity Purification of TST-Proteins using Strep-Tactin®XT 4Flow® high capacity in IMCStips® (Cook, et al. (2022). Application Note)

SPR

Application of Strep-Tactin®XT for affinity purification of Twin-Strep-tagged CB2, a G protein-coupled cannabinoid receptor (Yeliseev, et al. (2017). Protein Expression and Purification)
A conformation-specific nanobody targeting the nicotinamide mononucleotide-activated state of SARM1 (Hou, et al. (2022). Nature Communications)