Protein expression is a complex topic with many variables. Therefore, it is e.g. hard to predict whether a recombinant protein is expressed in a soluble form or whether it forms inclusion bodies or is partially degraded. To be prepared for the most common difficulties (see below) the attachment of two tags to a recombinant protein provides the ability of obtaining highly pure and homogenous protein preparations.
Important reasons for two different affinity tags on one protein:
- Purification of full length proteins
- Highest purification factors
- Using denaturing OR physiological purification conditions
- Optimizing purification protocols directly from the culture medium
A smart double tag pair is the combination of Strep-tag® and 6xHistidine-tag. Generally, it is recommended to attach one tag to the N-terminus and the other to the C-terminus.
Recombinant proteins containing a 6xHistidine-tag can be purified by Ni-NTA (nickel-nitrilotriacetic acid) chromatography which is based on the interaction between a transition Ni2+ ion immobilized on a matrix and the histidine side chains. Following washing of the matrix 6xHistidine-tag fusion proteins can be eluted by adding free imidazole or EDTA or by reducing the pH.
IBA offers several Ni-NTA resins, columns and cartridges for purification of 6xHistidine-tag proteins.
- One-step purification from crude lysates
- High binding affinity and high capacity
- Purification under native or denaturing conditions is possible
- Pre-charged, ready-to-use matrices for any scale of purification
Purification of full length proteins
Recombinant proteins may be partially degraded during expression, which cannot be prevented by adding protease inhibitors during downstream processing. Soluble degradation products still carrying the tag are co-purified and cause an inhomogeneous protein preparation with protein fragments of different lengths. This problem can be solved by adding a second tag to the other protein terminus. Performing a second purification run using the affinity of this second tag selects for full-length proteins.
Highest purification factors
Strep-tag® purification enables isolation of recombinant protein at over 99 % purity in one step under physiological conditions. However, impurities may arise, depending on the recombinant protein. Although the problem may be solved by changing the resin, the use of a second affinity tag may potentially be more efficient. To obtain reliable results in any downstream application, e.g. crystallography, immunizations or HTS, a highly pure product is essential.
Using denaturing OR physiological purification conditions
It is hard to predict whether a recombinant protein is expressed in soluble form or forms inclusion bodies. This depends, e.g. on the strength of the promoter used for expression and on the folding rates of the protein itself. If the protein is soluble and functional, Strep-tag® is the best choice because of its mild purification conditions keeping the protein bioactive. If the protein is insoluble and forms inclusion bodies after expression, it has to be solubilized with chaotropic salts, like guanidine or urea, at high concentrations in order to allow affinity chromatography. Purification under such conditions can be excellently achieved by using the metal chelate activity of the 6xHistidine-tag. Proteins can be purified and optionally refolded on Ni-NTA columns. If necessary, they can be further purified as refolded protein via Strep-tag® in physiological buffer.
Optimizing purification directly from the culture medium
A frequently applied expression strategy, for higher expression hosts in particular, is secretion of the recombinant protein to the
culture medium. In such cases a first capturing step with the 6xHistidine-tag
is recommended. Due to the high affinity of the 6xHistidine-tag to Ni-NTA
matrix, the recombinant protein can be efficiently collected even by performing
batch purification. As a positive side-effect, biotin, which is incompatible
with Strep-tag® purification and generally present in culture media, is
removed in this step. Thus, if necessary, a second purification step can be
performed using Strep-tag® leading to highly pure protein.
Fiedler M, Horn C, Bandtlow C, Schwab ME, Skerra A, 2002: Protein Eng. Nov;15(11):931-41. An engineered IN-1 F(ab) fragment with improved affinity for the Nogo-A axonal growth inhibitor permits immunochemical detection and shows enhanced neutralizing activity.