Double-tag proteinsProtein expression is a complex topic with many variables. Therefore, it is e.g. hard to predict whether a recombinant protein is expressed soluble or 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 flexibility to obtain a highly pure and homogenous protein preparation.
Purification of full length proteinsRecombinant 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 factorsStrep-tag¢ç purification enables isolation of recombinant protein at over 99% purity in one step under physiological conditions. However, depending on the recombinant protein, impurities may arise. Although the problem may be solved by changing the resin, the usage of a second affinity tag may potentially be more efficient. To obtain reliable results in any downstream application like, e.g., crystallography, immunizations or HTS, a highly pure product is essential. Using denaturing OR physiological purification conditionsOptimizing purification directly from the culture mediumEspecially for higher expression hosts (e.g. insect cells) an often applied expression strategy is the 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¢ç 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¢ç leading to highly pure protein. |
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