In most cases, functional tests can be performed using the intact histidine-tagged protein. If removal of the tag is necessary, then procedures similar to GST tag removal can be followed, that is, speciﬁc recognition sites are incorporated to allow subsequent enzymatic cleavage. The precise protocols required for cleavage and puriﬁcation will depend on the original vectors and the properties of the speciﬁc enzymes used for cleavage.
rTEV protease (Invitrogen) has a (histidine)6-tag and recognizes the amino acid sequence Glu-Asn-Leu-Tyr-Phe-Gln↓Gly. Glu, Tyr, Gln and Gly are needed for cleavage between the Gln and Gly residues (↓). N-terminal (histidine)6-tags can be removed. The advantage of this enzymatic cleavage is that the protein of interest can be repuriﬁed using the same Ni Sepharose medium or prepacked column. The (histidine)6-tag and the (histidine)6-tag rTEV protease will both bind to the column, and the protein of interest can be collected in the ﬂowthrough.
The amount of enzyme, temperature, and length of incubation required for complete digestion vary according to the speciﬁc tagged protein produced. Determine optimal conditions in preliminary experiments. Remove samples at various time points and analyze by SDS-PAGE to estimate the yield, purity, and extent of digestion. Approximate molecular weights for SDS-PAGE analysis:
|r TEV protease
C arboxypeptidase A*
| Mr 29 000
Mr 94 000
* for the removal of C-terminal (histidine)6-tags.
Some cleavage procedures will require a second puriﬁcation step to remove the protease or other contaminants. Conventional chromatographic separation techniques such as SEC (usually no need for optimization), IEX, or HIC will need to be developed (Appendix 11, Principles and standard conditions for different purification techniques).
On the following page we present an example of automated tag removal using ÄKTAxpress. All multistep puriﬁcation protocols in ÄKTAxpress can be combined with automated on-column tag cleavage. Tag cleavage is always performed on the afﬁnity column prior to further puriﬁcation steps. When the cleaved protein has been eluted, the afﬁnity column is regenerated and afﬁnity tag, tagged protease, and remaining uncleaved protein are collected in a separate outlet. The procedure involves binding the tagged protein, injection of protease, incubation, elution of cleaved protein, and collection in capillary loop(s), followed by further puriﬁcation steps.
The example in Figure 3.53 shows puriﬁcation results for a (histidine)6-tagged protein, APC234 (Mr 32 500), expressed in E. coli. The Mr of the cleaved product is 30 000. After harvest, cell lysis was performed by sonication. The samples were clariﬁed by centrifugation prior to sample loading.
AC, DS, IEX, and SEC were all performed on ÄKTAxpress using columns as indicated in the ﬁgure. The purity of each sample was analyzed by SDS-PAGE (Coomassie staining). The reduced samples were applied on an SDS-polyacrylamide gel. Approximately 7.5 µg of protein was loaded per lane.1
Figure 3.53.(A) Four-step protocol for puriﬁcation of (histidine)6-tagged protein cleaved with AcTEV protease. (B) SDS-PAGE analysis. The gel was stained with Coomassie.