Strep-Tactin®XT – a streptavidin mutant that allows reversible binding of biotinylated proteins
Biotinylation of proteins via Avi-tag is often used when protein immobilization for analytical applications is required. The binding to streptavidin is nearly irreversible and can only be dissolved by using harsh reagents such as guanidine HCl (GuHCl) or boiling. Since harsh elution conditions can affect the function of the target protein, the initial purification is usually done via a different affinity tag such as His-tag. However, the use of a second affinity tag can easily be circumvented by using Strep-Tactin®XT, a streptavidin mutant.
Strep-Tactin®XT was developed for binding Strep-tag®II and Twin-Strep-tag® with a high affinity. To achieve this, the binding pockets of streptavidin were mutated, enabling efficient binding of both Strep-tag variants. As a consequence of these mutations, the previous strong binding of biotin to streptavidin was reduced and allowed reversible binding of biotin and biotinylated proteins. This way, Strep-Tactin®XT is suitable for purifying proteins via a biotinylated Avi-tag, requiring only mild elution conditions and thereby keeping proteins in their functional state.
Figure 1. Strep-Tactin®XT binds to biotinylated proteins. Binding is reversed by addition of excess biotin.
One-step purification of biotinylated Avi-tag proteins with mild elution – no second affinity tag needed
The great advantage of using Strep-Tactin®XT for the purification of biotinylated Avi-tag proteins is the ease of use. After centrifugation, cell culture supernatants or cell lysates containing in vivo biotinylated proteins can directly be applied to a column containing Strep-Tactin®XT agarose beads. After washing, protein elution is done with biotin, a non-denaturing agent that does not affect protein function. This one-step purification procedure yields highly pure protein suitable for various downstream applications (Figure 2).
In this Application Note we show that
- Different in vivo biotinylated Avi-tag proteins are effectively enriched by Strep-Tactin®XT
- Purity remains high even for low concentrated samples
- Biotin contained in cell culture media does not affect purification efficiency
- Strep-Tactin®XT can be reused at least 10 times without loss in binding capacity
Ligand contamination caused by harsh elution conditions is avoided by using Strep-Tactin®XT for purifying biotinylated proteins
One problem of using harsh elution conditions such as GuHCl is not only the potential adverse effect on the target protein itself, but also on the ligand coupled to agarose beads. GuHCl can potentially cause leakage of the ligand from the resin (Figure 3A - right picture), causing sample contamination. This can be avoided by using Strep-Tactin®XT since mild elution with biotin yields the same amount of protein as harsh elution conditions, while sample purity is maintained (Figure 3A - left picture and 3B).
Figure 3: GuHCl elution can cause leaking of ligand from agarose beads causing impurities in the target protein sample (A), while protein yield is similar (B).
Small-scale purification of biotinylated proteins with magnetic beads
For applications such as expression screening or high-throughput purification of biotinylated proteins from limited sample volumes, magnetic bead-based affinity purification offers a robust and scalable approach. When using MagStrep® Strep-Tactin®XT beads, the majority of biotinylated proteins is already bound after 10 minutes incubation (Figure 4A). In total, around 10 µg/µl biotinylated Avi-tag protein can be purified by magnetic beads (Figure 4B). Strep-Tactin®XT is offered in both agarose and magnetic bead matrices, providing flexibility to adapt the purification strategy to specific throughput and automation requirements.
Figure 4: MagStrep® Strep-Tactin®XT beads bind to in vivo biotinylated Avi-tag GFP (GFP-Avi-bio) within 10 min (A), yielding 10 µg/µl protein (B).
Strep-Tactin®XT is a cost-effective & time-saving alternative to other resins that reversibly bind biotinylated proteins
Since purifying biotinylated proteins via streptavidin is potentially harmful for the protein of interest due to elution with GuHCl or boiling, resins that reversibly bind biotinylated proteins were developed. Compared to Strep-Tactin®XT, these resins have a 3-6x lower binding capacity (Figure 5A) and are also 2-3x more expensive than Strep-Tactin®XT resin (Figure 5B). In addition, Strep-Tactin®XT can be regenerated several times without loosing any binding capacity (Figure 5C), making it a cost-efficient and sustainable alternative to other commercially available reversible resins for biotinylated proteins.
Figure 5: Compared to competitors that offer resin that reversibly bind biotinylate proteins, Strep-Tactin®XT has a 3-6x higher binding capacity (A) and is 2-3x more cost effective (B). In addition, multiple regenerations without loss in binding capacity are possible (C).
One way to ensure mild elution of biotinylated proteins from agarose beads is the use of monomeric avidin instead of streptavidin. However, this method requires conditioning of the resin prior to use, meaning that non-reversible binding sites have to be blocked first. This additional step is not necessary when using Strep-Tactin®XT. The columns can directly be used for purifying biotinylated proteins without any initial preparation steps. Hence, Strep-Tactin®XT is an easy to use and time-saving option for biotinylated protein purification.
Figure 6: Compared to monomeric Avidin-based resins that reversibly bind biotinylated proteins, Strep-Tactin®XT does not require any pre-conditioning, making the purification procedure simple and time-efficient.
