Strep-tag purification uses a short peptide tag (Strep-tag II) that binds specifically to engineered streptavidin (Strep-Tactin) for gentle, one-step protein purification under physiological conditions without metal ions or reducing agents.
Principle
The Strep-tag II is an eight-amino-acid peptide sequence (WSHPQFEK) that mimics biotin’s interaction with streptavidin. It binds Strep-Tactin, an engineered variant of streptavidin with a modified binding pocket that accommodates the peptide with high affinity (K_d ≈ 1 µM). Binding is reversible: elution uses 2.5 mM desthiobiotin, a biotin analog that competitively displaces the tag. Desthiobiotin binds more weakly than biotin and is removed during buffer exchange, allowing resin regeneration. Alternatively, 10 mM biotin can be used for complete elution but prevents resin reuse. The entire procedure is compatible with physiological buffers, preserving native protein structure and activity.
Twin-Strep-Tag
The Twin-Strep-tag (two Strep-tag II sequences in tandem separated by a short linker) dramatically improves binding affinity (K_d ≈ 10⁻¹¹ M), enabling capture of proteins expressed at very low levels and purification from dilute samples. It also allows quantitative capture of protein complexes. The Twin-Strep-tag is recommended for challenging targets and for affinity capture of protein interaction partners from endogenous expression levels.
Resins and Formats
Strep-Tactin agarose (IBA Lifesciences, now part of Cube Biotech) offers binding capacity of approximately 50 nmol Strep-tag II peptide per mL (≈1.3 mg of a 26 kDa protein). Strep-Tactin Superflow high-performance resin is compatible with FPLC systems at flow rates up to 5 mL/min. Strep-Tactin magnetic beads are available for small-scale purification and pull-down experiments. Strep-TactinXT is an improved version with higher affinity for the conventional Strep-tag II, eliminating the need for the larger Twin-Strep tag in most applications.
Buffers
Loading/wash buffer: 100 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA. Elution buffer: loading buffer plus 2.5 mM desthiobiotin. Regeneration buffer: 1 mM HABA (hydroxyazophenyl-benzoic acid) or 10 mM biotin in loading buffer to strip residual bound protein or desthiobiotin. The buffer is compatible with reducing agents (< 2 mM DTT or TCEP) and mild detergents (0.1% Triton X-100, 0.05% NP-40). Strong reducing agents (> 10 mM DTT) and biotin-containing solutions (> 2 µM) should be avoided in the loading step as they interfere with binding.
Protocol
Equilibrate Strep-Tactin resin with loading buffer. Load clarified lysate (batch incubation 30 min, 4 °C, or column at 0.5–1 mL/min). Wash with 10–20 column volumes of loading buffer. Elute with 6–10 column volumes of elution buffer (desthiobiotin). If using biotin, elute with 5 column volumes and note that the resin cannot be reused. Regenerate with HABA or biotin buffer, then equilibrate for reuse up to five times. Collect fractions and analyze by SDS-PAGE.
Advantages
The Strep-tag is small (8 amino acids, 1 kDa) and rarely affects protein function, making tag removal unnecessary for most applications. Elution with desthiobiotin under physiological conditions preserves enzymatic activity, complex integrity, and antibody binding. The system is compatible with His-tag for dual-tag purifications. The absence of metal ions eliminates the risk of metal-catalyzed oxidation. Applications include recombinant protein purification, protein complex isolation, and pull-down of interaction partners.
