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Introduction
Recombinant fusion tags aid protein expression studies by providing methods for purification and detection when protein-specific antibodies are not available. Histidine-containing tags used for immobilized metal affinity chromatography (IMAC) are the most commonly utilized system for protein purification, but can lack specificity when used for immunodetection. Conversely, the greater specificity of an antibody-based tag, such as FLAG®, is better suited for high sensitivity immunodetection.
In this work, we demonstrate the Metal Affinity Tag (MAT-Tag), a complete and versatile system for the expression, purification, and detection of recombinant fusion proteins. Many expression vectors included in the MAT-Tag System contain the FLAG epitope tag, allowing for IMAC purification coupled with FLAG detection.
Methods and Discussion
The MAT-Tag System
The system, incorporating vectors, purification resins and detection antibodies, utilizes a seven amino acid histidine-based affinity tag (N-His-Asn-His-Arg-His-Lys-His). Purification of target fusion proteins is achieved by conducting a single IMAC step.
Western Blot Immunostaining with ANTI-FLAG®/Anti-MAT monoclonal antibodies
ANTI-FLAG and Anti-MAT monoclonal antibodies specifically recognized the full-length FLAG-BAP-MAT fusion protein by immunostaining of Western blots of samples from induced E. coli cell lysates (Fig. 1, lanes 3 and 6). No protein was detected in samples of lysates of uninduced cells (Fig. 1, lanes 2 and 5).
Figure 1. (Click to enlarge)
Purification of FLAG-GrpE-MAT by IMAC
GrpE was cloned into an expression vector to generate an N-terminal FLAG and a C-terminal MAT fusion protein. FLAG-GrpE-MAT fusion protein expression was induced in E. coli, and the fusion protein was purified from the cell lysate on a 1 ml HIS-Select® HF (high flow) Nickel Affinity Gel column. The FLAG-GrpE-MAT purification was analyzed by SDS-PAGE analysis of purification fractions (Figure 2).
Figure 2. (Click to enlarge)
Conclusion
Compatible with IMAC and with the sensitivity of antibody-specific detection, the utility of the MAT-Tag system provides investigators greater flexibility when expressing recombinant proteins. Sigma-Aldrich offers the MAT-Tag product line that includes FLAG-MAT-fusion protein expression vectors for bacterial and mammalian systems and complimentary Anti-MAT or ANTI-FLAG monoclonal antibodies for downstream detection.
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