Isolation, Characterization, and Imaging Analysis of Complexes Using HaloTag Technology. Michael Ford, Richard Jones, Ravi Amunugama, David Allen MS Bioworks, 3950 Varsity Drive, Ann Arbor, MI, U.S.A. 48108 Jacqui Mendez, Nancy Murphy, Marie Schwinn, Hélène Benink, Danette L. Daniels, and Marjeta Urh Promega Corporation, 2800 Woods Hollow Road, Madison, WI U.S.A. 53711

Abstract and introduction Mediator isolation using MED26-Halo and Halo-MED13 Highly specific and distinct Mediator complex capture

Expression and pulldown The understanding of complex assembly and mapping of protein Halo-Mediator fusions in HEK293 Mass Spectrometry Analysis NSAF Heat Map of Mediator Subunit Coverage based upon Mediator Domains interactions has rapidly grown in recent years due to significant MED-HT HaloTag • Run pulldown samples on gel advances in mass spectrometry. As experiments turn toward Vector MED26-HaloTag HaloTag-MED13 MED • Excise into 10 segments per Head Domain Middle Tail Unassigned Kinase MED HT HT MED characterization of the human proteome and the complexity it presents, a Transfection 26 Ctrl Ctrl 13 sample. MED MED MED MED MED MED MED MED MED MED MED MED MED MED MED MED MED MED MED MED MED MED MED MED MED MED MED MED CDK Cycl need remains to efficiently capture complexes intact, particularly weak or • Trypsin digestion 6 8 11 17 18 19 20 22 28 29 30 1 4 7 9 10 21 14 15 16 23 24 25 26 27 31 12 13 8 C 220 220 MED26 transient interactors as well as large multiprotein complexes. Here we 120 • Nano LC-MS/MS (LTQ Orbitrap Lysis, 120 H 100 100 Capture MED13 present a new technology based upon the use of a protein fusion tag, T 80 80 Velos) 60 HaloLink 60 • Database search (Mascot) 50 termed HaloTag, which allows for highly specific and covalent Resin 50 HT 40 • Database processing (Scaffold) Heat immobilization of complexes, as well as the ability to do 40 NSAF 30 Map SDS 30 • Data Analysis (Spectral Counting Values .05-1E-04 1-5E-04 0.5-1E-03 1-5E-03 0.5-1E-02 correlative cellular localization studies using fluorescent ligands. In Elution and Normalized Spectral Key Silver Stain Silver Stain studies presented here, this technology was used to further Abundance Factors (NSAFs)) understanding of the multi-protein Mediator complex assemblies and their cellular function. Significant enrichment over control of proteins from both HaloTag constructs. Kinase module members excluded from core Mediator complexes containing MED26. Successful expression and capture of MED13 (239kDa) as a HaloTag fusion. MED26 excluded from Mediator-Kinase module complexes.

HaloTag technology NSAF plot of Mediator subunits from MED26 Isolations Correlative cellular localization studies of Mediator

Cellular imaging of HaloTag fusion protein HaloTag ligands and applications 6E-03 HaloTag Mediator fusions MED26-Halo Halo-MED13 Protein capture and display Functional HaloTag surface ligands 5E-03 Protein of O Interest HT + Cl O group Protein display Protein purification 4E-03 H Covalently Interaction analysis binds to T Label chloroalkane HaloTag fuisons ligands with TMR Protein labeling and detection 3E-03 HaloTag fluorescent ligands Protein of Functional Functional O NSAF Averaged HT O Cellular imaging Interest group group 2E-03 Gel analysis Quantitation HaloTag reactive ligands 1E-03 Attach modifications Attach to particles 0E+00 R MED4 MED20 MED31 MED22 MED1 MED29 MED14 MED17 MED11 MED6 MED18 MED16 MED24 MED15 MED30 MED28 MED27 MED23 MED8 MED7 MED10 MED19 MED25 MED9 MED21 Attach to surfaces MED26-Halo 5E-03 5E-03 4E-03 3E-03 3E-03 3E-03 3E-03 3E-03 2E-03 2E-03 2E-03 2E-03 2E-03 2E-03 2E-03 2E-03 1E-03 1E-03 1E-03 8E-04 7E-04 6E-04 6E-04 5E-04 4E-04 Halo-Ctrl 0000000000000000000000000 HaloTag (HT) is a genetically encoded 33kDa protein fusion tag. Excellent enrichment and capture of core Mediator subunits in biological triplicates. Specific and restricted nuclear localization of HaloTag Mediator fusion Engineered to covalently bind various ligands, imparting multi-functionality. No Mediator subunits identified in HaloTag alone control.

Mediator Transcriptional Co-activator and Design NSAF plot of Mediator subunits from MED13 Isolations Summary

1.0E-02 HaloTag technology allows for broad study of protein function both in vitro and in vivo, including complex isolation and cellular imaging. 70S Ribosome 8.0E-03

Rapid and covalent isolation of complexes promotes maintenance of complex 6.0E-03 integrity, particularly macromolecular complexes.

HaloTag TEV Site 4.0E-03 Efficient isolation of core Mediator and Mediator bound to it is kinase module Averaged NSAF Averaged using MED26-HaloTag and Halo-MED13, respectively.

2.0E-03 Mass spectrometry analysis showed distinct complexes associated with Knuesel M T et al. Dev. 2009;23:439-451 different Mediator subunits. 0.0E+00 Cyclin MED20 MED27 MED17 MED4 MED12 MED31 MED30 MED14 MED23 MED18 MED24 MED6 MED1 CDK8 MED22 MED15 MED16 MED29 CDK19 MED28 MED8 MED10 MED25 MED11 MED7 C Halo-MED13 7E-03 4E-03 4E-03 4E-03 4E-03 4E-03 3E-03 3E-03 3E-03 3E-03 3E-03 3E-03 3E-03 3E-03 3E-03 2E-03 2E-03 2E-03 1E-03 1E-03 1E-03 1E-03 9E-04 8E-04 5E-04 5E-04 Halo-Ctrl 00000000000000000000000000 Cellular imaging revealed discrete nuclear localization of MED26 and MED13. Mediator exists in two forms: Core Mediator and Mediator+Kinase Module Excellent enrichment and capture of core Mediator subunit is biological duplicates. Place HaloTag on Core component MED26, and Kinase Module component, MED13 Additional capture of CDK8 Module subunits (CDK8, Cyclin C, and MED12) www.promega.com www.msbioworks.com