Benefits of Strep-tag® vs His-tag purification
Both, Strep-tag® and His-tag, are widely used tools for affinity purification via a peptide tag. But when it comes to purity and versatility in applications, some differences between both systems are obviously.
Especially, the newly engineered Strep-Tactin®XT in combination with the Twin-Strep-tag® (alternatively with Strep-tag®II) has an improved binding affinity which enables a broad variety of different applications:
- Batch purification
- Purification under denaturing conditions
- Membrane proteins
- Purification of low abundant proteins
- Assay applications
Read why the Strep-tag® system is the superior purification tool:
+ high purity, even under denaturing conditions and in batch purification
+ no additional purification necessary
+ physiological buffer conditions
+ mild reversibility without changing the overall buffer conditions
+ purification of metallo-proteins
+ beneficial for membrane proteins
+ SPR and other high affinity applications
- less yield compared to His-tag
+ resin can be recharged
+ denaturing conditions with Gua-HCl
- not suitable for metal proteins
- interacts unspecifically with complex forming amino acids
- does not tolerate chelating agents
- works only best for E. coli expression (often problematic after mammalia expression)
- time consuming elution gradient for higher purity
Download detailed experimental data:
Shown is the comparison of two randomly chosen proteins, which were either fused to 6xHis-tag or Twin-Strep-tag®. Proteins were purified via Ni-NTA resin, which was done under optimal His-tag purification conditions, or via Strep-Tactin®XT resin using physiological buffer conditions on the right side, respectively. Both proteins show low background when purified using the Strep-tag® system.
Purification under denaturing conditions
The expression of recombinant proteins, especially using bacterial vectors and hosts, is a mature and powerful technology and allows the production of a desired protein in large quantities. The procedure to obtain a recombinant protein is straightforward. The gene of interest is cloned into the appropriate expression vector, transformed in the host of choice, induced and then the expressed protein can be purified. In practice, however, things turn out to be more difficult.
One problem is, of course, how to isolate it in an active form.
Soluble proteins can be recovered with good yields (> 50 %), and insoluble proteins, which must undergo a denaturation and folding cycle, can be recovered with more modest yields (5 % to 20 %). [Wingfield, 2016; Rosano and Ceccarelli, 2014]
IBA’s newly developed Strep-Tactin®XT with its near covalent binding affinity for Twin-Strep-tag® (and improved binding affinity for Strep-tag®II) enables increased protein yields of insoluble proteins when purified with up to 6 M urea.
The main advantages of the Strep-tag® system compared to His-tag purification are:
- purification within a single purification step – no additional chromatography step is required
- protein purity < 95 %
Strep-tag® system vs His-tag system
The 3rd generation Strep-tag® system can now be used for purification under denaturing conditions using up to 6 M urea.
Check the application note for a comparison of Strep-tag® system III to His-tag:NiNTA purification under denaturing conditions.
Download experimental data: