Recombinant Protein Expression, Purification And Detection

Ned Watson, Rebecca L. Davis, Michael Scott, Greg Davis, Efrat Reem, Eliezer Kopf, Dorit Zharhary, Richard J. Mehigh and William K. Kappel

Introduction
Polypeptide epitope tags fused to recombinant proteins facilitate studies of protein expression and function by providing simple methods for purification and detection of fusion proteins when target protein-specific antibodies are not available. Histidine-containing tags, developed for immobilized metal affinity chromatography (IMAC), are the most commonly utilized affinity tags for one-step purification of recombinant proteins, but can lack specificity when used for immunodetection. Conversely, the greater specificity of an antibody-based tag, such as FLAG Sigma-Aldrich, St. Louis, MO), is better suited for specific and high sensitivity immunodetection.

Figure 1. Purification of target fusion proteins
In this work, we demonstrate utility of the recently developed Metal Affinity Tag (MAT), MAT-Tag vectors and Anti-MAT-Tag monoclonal antibody as a complete and versatile system for expression, purification and detection of recombinant proteins. Many expression vectors developed for the MAT-Tag system also contain the FLAG epitope tag, allowing for IMAC purification to be coupled with FLAG tag detection.

Materials and methods
Reagents
(All reagents are from Sigma-Aldrich unless otherwise specified.) The Anti-MAT monoclonal antibody was purified from mouse ascites fluid generated by a hybridoma (MAT 1-87) that was produced by fusion of NS1 mouse myeloma cells and splenocytes from BALB/c mice immunized with synthetic MAT peptide (HNHRHKHGGGC) conjugated to KLH via the C-terminal cysteine.

Expression and purification of MAT-tagged proteins
The coding regions for GrpE, bacterial alkaline phosphatase (BAP) and turboGFP (Evrogen, Moscow, Russia) were cloned into an E. coli expression plasmid, pFLAG-MAC, with an N-terminal FLAG tag and a C-terminal MAT tag. The proteins were expressed in E. coli strain BL21 after induction with IPTG. The induced cells were collected by centrifugation and lysed in CelLytic B lysis reagent. After centrifugation of the lysates, the supernatant fractions were subjected to IMAC affinity purification on 1 ml HIS-Select HF Affinity Gel columns. The eluted fractions were analyzed by SDS-PAGE. Fractions containing the purified target proteins were dialyzed and stored in 20% glycerol at 㪬?C.

Western blot immunostaining analysis FLAG-BAP-MAT fusion protein expression

Lysates from uninduced and induced E. coli cultures and ColorBurst Markers were separated by SDS-PAGE and blotted to nitrocellulose. After blocking, the blot was immunostained using either ANTI-FLAG? M2-HRP conjugate or Anti-MAT monoclonal antibody (0.5 μg/ml) followed by Rabbit Anti-Mouse IgG-HRP conjugate. The blots were developed and visualized with colorimetric TMB substrate.

Immunoaffinity pull-down of MAT tagged GFP from a mammalian cell lysate
Figure 2. Western blot analysis of a FLAG-BAP-MAT fusion protein in lysates

The purified turboGFP (27 kD) fusion protein with an N-terminal FLAG-tag and a C-terminal MAT-tag was either spiked into a COS-7 cell lysate (107 cells in 1 ml RIPA buffer) at 25 mg/ml (+) or not spiked (-) and captured using Anti-MAT monoclonal antibody and EZview Red Protein G Affinity Gel. After washing the affinity gel samples, the bound proteins were eluted and analyzed by SDS-PAGE with EZBlue staining.

Immunostaining MAT-tagged MAP kinase expressed in mammalian cells
Adherent HEK293 cells were transfected with a FLAG-MAT-MAPK expression vector. After two days incubation the cells were fixed with 3% paraformaldehyde and 0.5% Triton X-100. The fixed cells were stained with 5 mg/ml Anti-MAT monoclonal antibody and developed with Anti-Mouse IgG (Fab specific)-FITC conjugate at a 1:40 dilution. The cells were visualized by fluorescence microscopy and photographed.

Results
The MAT tag system utilizes a proprietary seven amino acid histidine-based epitope affinity tag (N-His-Asn-His-Arg-His-Lys-His). The system includes expression vectors, IMAC purification resins and a tag specific monoclonal antibody. The system is suitable for large-scale purification, sensitive and specific detection, and small-scale immunoaffinity capture of MAT-tagged fusion proteins.

Rapid and specific purification of target fusion proteins is readily achieved by a single IMAC affinity chromatography step, as demonstrated in Figure 1. The coding sequence for the E. coli GrpE protein was cloned into an expression vector to generate an N-terminal FLAG and a C-terminal MAT tagged fusion protein. Expression of the FLAG-GrpE-MAT fusion protein was induced in E. coli, and the MAT-tagged protein was purified from the cell lysate on a 1 ml HIS-Select HF (high flow) Nickel Affinity Gel column. Fractions from the FLAG-GrpE-MAT purification were analyzed by SDS-PAGE (Figure 1). About 7 mg of purified protein was recovered from the elution fractions from a 500 ml culture (Figure 1, lanes 9 and 10).

Figure 3. Capture of MAT-tagged protein from a mammalian cell lysate.
Differentially tagging both the N- and C-termini can be useful for identifying full length expressed fusion protein, as well as truncated fragments from the target protein that might result from proteolytic action or from pre-mature transcription or translation termination. Western blot analysis of a FLAG-BAP-MAT fusion protein in lysates from uninduced and induced E. coli cells was performed after SDS-PAGE separation of the lysate proteins. Immunostaining of duplicate portions of the blot with an ANTI-FLAG and an Anti-MAT monoclonal antibody, respectively, readily revealed the full-length fusion protein, as well as different N- or C-terminally tagged fragments that reacted with only one of the tag specific antibodies (Figure 2, lanes 3 and 6). In addition, no protein bands were detected in samples of lysates from uninduced cells, demonstrating the high specificity of the two tag-specific antibodies employed (Figure 2, lanes 2 and 5). The signal on the immunostained blot using the Anti-MAT monoclonal antibody was comparable to the signal generated by the ANTI-FLAG M2 monoclonal antibody, demonstrating comparable sensitivity of target protein detection for Western blotting applications.

To demonstrate the utility of the Anti-MAT antibody for immunoaffinity capture (immunoprecipitation) of a target protein from a complex mixture, we tested the ability of the antibody to capture specifically a MAT-tagged protein from a mammalian cell lysate. Purified turboGFP (27 kD) with an N-terminal FLAG tag and a C-terminal MAT tag (Figure 3, lane 2) was spiked into a COS-7 cell lysate (Figure 3, lane 3) and immunoprecipitated as described in the legend for Figure 3. The protein captured during the immunoprecipitation was analyzed by SDS-PAGE. The Anti-MAT antibody specifically captured the 27 kD MAT-tagged GFP target protein with no cross-reactive protein contaminants detectable (Figure 3, lanes 4 and 5).

We also tested the ability of the Anti-MAT monoclonal antibody to detect MAT-tagged fusion proteins in mammalian cells by immunostaining. MAT-Tagged MAP kinase (MAPK) was expressed in HEK293T cells after transfection with an expression construct. After two days the cells were fixed, permeabilized and immunostained with the Anti-MAT monoclonal antibody. The expression of the MAT-tagged MAPK was readily detected in the cytoplasm and the nuclei of transfected cells (Figure 4). Mock transfected cells showed no signal (data not shown).

Conclusions
Figure 4. Detection of MAT-tagged fusion proteins in mammalian cells by immunostaining.
A new metal affinity tag, the MAT tag, along with MAT-fusion protein expression vectors and the Anti-MAT monoclonal antibody, forms a complete protein expression system. It has demonstrated utility for expression, purification, and detection of a variety of recombinant fusion proteins. Combining the simplicity of one-step IMAC affinity purification with the sensitivity of epitope tag-specific antibody detection provides investigators with flexibility when expressing recombinant proteins. In addition, the availability of dual FLAG and MAT tag vectors for bacterial and mammalian expression systems, with the complimentary ANTI-FLAG and Anti-MAT monoclonal antibodies, allows more detailed and flexible detection and analysis of the expressed fusion proteins.

About the authors
Ned Watson, Rebecca L. Davis, Michael Scott, Greg Davis, Efrat Reem, Eliezer Kopf, Dorit Zharhary, Richard J. Mehigh and William K. Kappel are with Sigma-Aldrich in St. Louis, Missouri.

More information on protein expression, purification and detection and the methods discussed in this paper is available from:
Sigma-Aldrich
800-325-3010
www.sigmaaldrich.com

References
1. Kolodziej, P. A. and Young, R. A. Epitope tagging and protein surveillance. Methods Enzymol. 194:508-519 (1991).
2. Porath, J. Immobilized metal ion affinity chromatography. Protein Expr. Purif. 3:263-281 (1992).
3. Street, G. (Ed.), Highly Selective Separations in Biotechnology. Chapman and Hall, London (1994).
4. Harlow, E. and Lane, D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York p. 423-470 (1988).

 

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