Focus: Vector Construction How to Construct and Use a HaloTag® Coding Region Control Expression Vector The HaloTag® 7 Flexi® Vectors offer the convenience of expressing fusion proteins to study protein structure and function in a variety of expression systems. Here we describe a way to make a HaloTag ®-only control expression vector that expresses only the HaloTag® 7 protein, and highlight a few applications where this construct can be useful. By Shoba Ananthanarayanan, M.S., Robin Hurst, M.S., Jim Hartnett, M.S., Michael Slater, Ph.D., and Trista Schagat, Ph.D. Promega Corporation Published in April 2009 Introduction It is common to express gene products as fusion or tagged proteins to elucidate numerous cellular interactions. The HaloTag® 7 protein, a multifunctional tag, has been genetically modified to covalently bind its ligand, making the expressed peptide useful as a protein immobilization (1) or a labeling tag (2). When using the HaloTag ® 7 coding region as a protein immobilization tag for applications such as protein arrays (3), pull downs (4) and chromatin immunoprecipitation (ChIP) assays (5) or as a labeling tag [e.g., imaging studies (6,7) and fluorescent gels (8)], it can be helpful to have a construct that can express only the HaloTag® protein for controls. Here we describe the design of a HaloTag ®-only control expression vector and discuss experimental scenarios where having a HaloTag® control construct enhances data quality and facilitates interpretation of results. Materials and Methods Design of a HaloTag® Control Expression Construct in N-terminal Fusion Vectors The Flexi® Vectors provide the option of expressing the HaloTag® coding region either as an N-terminal or a C-terminal partner of the fusion protein (9). In the N-terminal vectors, the fusion protein is cloned downstream of the HaloTag ® coding region, replacing the lethal gene, barnase. Hence there is no stop codon in the HaloTag® sequence. To express only the HaloTag® protein, it is necessary to both remove the barnase gene and introduce a stop codon for the HaloTag ® coding region. This can be done by designing and annealing oligos that contain a stop codon (e.g., TAA) and replace barnase in the vector. This results in the expression of HaloTag® protein without cell death. A schematic of the HaloTag®-only control expression construct created using an oligo-based cloning strategy is illustrated below (Figure 1). This construct has all the elements to serve as a HaloTag ®-only expression control. In this example, the oligos have also been designed to recreate the SgfI and PmeI restriction sites. Thus, the construct could also be used as a cloning vector for future fusion expression projects requiring N-terminal HaloTag® fusions. Figure 1. Schematic of the HaloTag® control expression construct from N-terminal Flexi® Vectors using the oligo-design strategy. N-terminal HaloTag® vectors such as pFN19A (Cat.# G1891) and pFN19K (Cat.# G1841) (HaloTag® 7) T7 SP6 Flexi® Vectors contain the lethal barnase gene downstream of the HaloTag ® coding region. HaloTag®-only control constructs can be made by designing oligos that carry a stop codon and ends compatible for ligation. The vector is digested with SgfI and PmeI to remove the barnase gene. In a separate reaction, the oligos are annealed. The digested vector and the annealed oligos are ligated using T4 DNA Ligase (Cat.# M1794). An example protocol for creating a HaloTag®-only control expression vector with an Nterminal Flexi® Vector is presented below. Materials Required N-terminal HaloTag® 7 Flexi® Vector [e.g., pFN19A (HaloTag® 7) T7 SP6 Flexi® Vector (Cat.# G1891)] Flexi® Digest Buffer, 5X (Supplied as part of Cat.# R1851) Flexi® Enzyme Blend (SgfI and PmeI; Cat.# R1851) 1. Assemble the following reaction components to digest. Components Volume 5X Flexi® Digest Buffer 4µl Flexi® Vector (100ng) 1µl Flexi® Enzyme Blend (SgfI and PmeI) 2μl Nuclease-Free Water to a final volume of 20μl 2. Incubate the reaction at 37°C for 20 minutes. 3. Heat the reaction at 65°C for 20 minutes to inactivate the restriction enzymes. Store the reaction on ice until the ligation reaction. 4. Anneal the oligos: o Top strand: 5´ OH CGCGTAAGGGTAGGTTT (OD of 1 = 5.356fmol/ml) o Bottom strand: 5´ OH AAACCTACCCTTACGCGAT (OD of 1 = 4.948fmol/ml) Heat the oligos (50fmol/µl) in TE buffer [10mM Tris-HCl (pH 8.0); 1mM EDTA•Na2] to 95ºC, then slowly cool to room temperature for 15–30 minutes. 5. Ligate vector with T4 DNA ligase. Assemble the following ligation reaction components: Components Volume 2X Flexi® Ligase Buffer 10µl Digested DNA from Step 1 (~50ng total) 9µl Annealed oligos (50fmol/μl each) 1μl T4 DNA Ligase (Cat.# M1794; 20u/μl) 1μl Total Volume 21μl 6. Incubate reaction at room temperature for 1 hour. 7. Transform 1–2µl of the ligation reaction into high-efficiency, E.coli competent cells [e.g., JM109 Competent Cells (Cat # L2001)]. 8. Plate reaction and screen colonies on an appropriate antibiotic selection plate (e.g., LB-ampicillin or LBkanamycin). 9. Confirm expression of the HaloTag® protein, which has a molecular weight of approximately 32,000 Daltons, by labeling with the fluorescent HaloTag® TMR Ligand (Cat.# G8251) and subsequently visualizing on an SDS polyacrylamide-gel. (See the HaloLink™ Resin Technical Manual #TM250 or the HaloLink™ Magnetic Beads Technical Manual #TM291 for more details). Note: Any N-terminal HaloTag® 7 Flexi® Vector can be used to make a HaloTag®-only expression vector. Applications Controls in an experiment ensure consistency and validity of results. Having the right control is a critical attribute of solid experimental design. The HaloTag®-only control expression vector can serve as a good control for protein immobilization and labeling experiments. Below, we discuss the different applications that would benefit from expression of a HaloTag ®only control vector. Protein Immobilization Protein arrays, pull downs and ChIP assays: Recognition specificity between proteins and their interaction partners is essential when collecting experimental evidence via protein:protein binding experiments. To ensure that the prey protein is binding to the bait protein and not the HaloTag® moeity, it is beneficial to use the HaloTag® protein alone as a negative or background control. Fluorescent Protein Labeling Immunostaining and in vivo imaging experiments: Transfection of a control construct and the fusion construct of interest in intercellular (10) and intracellular trafficking experiments have been used to show trafficking was mediated by the fusion partner rather than nonspecific trafficking by the tag protein. HaloTag®-only expression controls can be used in such scenario to demonstrate protein-specific trafficking. Gel analysis and Western blotting: Fluorescent labeling of the HaloTag® 7 coding region can be used as a positive control for protein expression and labeling (see TM250 or TM291). If a fluoroimager for scanning the gel is unavailable, then perform Western blotting using the Anti-HaloTag® pAb (Cat.# G9281) to detect HaloTag® fusion proteins. In cases where the protein of interest is not detected, a positive control used in parallel with the fusion construct can be used to troubleshoot problems associated with protein expression or the blotting procedure. Transfections: Optimizing transfection efficiency is critical for controling variability in experimental outcome. Using a HaloTag®-only control expression construct in transfections prior to the experimental vector is a simple and efficient way of determining and optimizing transfection efficiency. Conclusion Controls are required in any experiment to account for differences that arise due to unknown biases present in the biological system of interest. We have shown that the HaloTag®-only control expression construct can serve as positive and negative control in a variety of experiments. Such a control construct adds to the options that N-terminal HaloTag® 7 Flexi® Vectors provide. References 1. Urh, M. et al. (2006) HaloLink™ Resin: High capacity, specificity and scalable throughput for protein analysis. Promega Notes 92, 24–9. 2. Los, G.V. et al. (2005) HaloTag™ Interchangeable Labeling Technology for Cell Imaging and Protein Capture. Cell Notes 11, 2–6 3. Nath, N. et al. (2008) Improving protein array performance: Focus on washing and storage conditions. J. Proteome Res. 7, 4475–82. 4. Urh, M. et al. (2006) HaloLink™ Resin For protein pull-down and analysis. Cell Notes 14, 15–9. 5. Hartzell, D.D. et al. (2007) HaloCHIP™ System: Mapping intracellular protein:DNA interactions using HaloTag® Technology. Promega Notes 97, 18–21. 6. Svendsen, S. et al. (2007) HaloTag® Protein: A novel reporter protein for human neural stem cells. Promega Notes 95, 20–2. 7. Los, G.V. et al. (2008) HaloTag: A novel protein labeling technology for cell imaging and protein analysis. ACS Chem. Biol. 3, 373–82. 8. Los, G.V. et al. (2005) HaloTag™ Interchangeable Labeling Technology for Cell Imaging, Protein Capture and Immobilization Promega Notes 89, 2–6. 9. McCornack, M., Schagat, T. and Slater, M. (2008) Expression of fusion proteins: How to get started with the HaloTag® Technology. Promega Notes 100, 13–5. 10. Elliott, G. and O’Hare, P. (1999) Intercellular trafficking of VP22-GFP fusion proteins. Gene Therapy 6, 149–51. Products may be covered by pending or issued patents or may have certain limitations on use. Please visit our patent and trademark web page for more information. Flexi and HaloTag are registered trademarks of Promega Corporation. HaloCHIP and HaloLink are trademarks of Promega Corporation.