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Abstract

Introduction

Protocol

Representative Results

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Acknowledgements

Materials

References

Biology

Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method

Published: July 27th, 2016

DOI:

10.3791/54389

1Department of Biochemistry, Brandeis University, 2Winship Cancer Institute, Emory University School of Medicine

The Tandem Affinity Purification (TAP) method has been used extensively to isolate native complexes from cellular extract, primarily eukaryotic, for proteomics. Here, we present a TAP method protocol optimized for purification of native complexes for structural studies.

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree of compositional heterogeneity of a complex do not usually impede progress in conducting such studies. In contrast, a complex intended for structural characterization should be purified in a state that is both compositionally and structurally homogeneous as well as at a higher concentration than required for proteomics. Recently, there have been significant advances in the application of electron microscopy for structure determination of large macromolecular complexes. This has heightened interest in approaches to purify native complexes of sufficient quality and quantity for structural determination by electron microscopy. The Tandem Affinity Purification (TAP) method has been optimized to extract and purify an 18-subunit, ~ 0.8 MDa ribonucleoprotein assembly from budding yeast (Saccharomyces cerevisiae) suitable for negative stain and electron cryo microscopy. Herein is detailed the modifications made to the TAP method, the rationale for making these changes, and the approaches taken to assay for a compositionally and structurally homogeneous complex.

Many major cellular processes are carried out by large protein and protein-RNA complexes1. A significant bottleneck to conducting biophysical and structural studies of such complexes is obtaining them of a suitable quality (i.e., homogeneity) and at an appropriate concentration. Isolating a complex from a native source has many advantages, including retaining relevant post-transcriptional and/or translational modifications of subunits and insuring proper complex assembly. However, large cellular complexes are often present in a cell at a low copy number and the purification must be highly efficient and occur under near physiological conditions to e....

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Note: The following protocol was devised for purification of a complex from 4 L of cell culture, approximately 40 g wet weight of cells. Once prepared, all buffers should be stored at 4 °C and used within a month of their preparation. Reducing agent and protease inhibitors are only added to buffers just prior to use.

1. Preparation of Whole Cell Extract for Tandem Affinity Purification 

  1. Growth of S. cerevisiae Cells
    1. Streak the desired TAP tagged S. ce.......

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A modified TAP method was used to purify from S. cerevisiae the U1 snRNP, an 18-subunit ribonucleoprotein complex. An initial TAP purification of the complex following the published protocol2,3 yielded a complex that appeared heterogeneous, migrating as three bands on a silver stained native polyacrylamide gel (Figure 2A). Multiple rounds of optimization of the TAP method, yielded a complex that migrated as primarily a single band on a native gel indic.......

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The TAP method utilizes two tags that balance a need for tight and selective binding to an affinity resin with a desire to maintain near physiological solution conditions. This balance serves to preserve stable interaction(s) of the tagged protein with interacting factor(s) for post-purification characterization. In addition, individual TAP tagged ORFs are available from a commercial source, so that one can obtain any yeast strain with a tagged protein for any yeast complex. Preserving the integrity of a complex and the .......

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The authors are grateful for the support and advice of Nikolaus Grigorieff. We thank Anna Loveland, Axel Brilot, Chen Xu, and Mike Rigney for helpful discussions and EM guidance. This work was funded by the National Science Foundation, Award No. 1157892. The Brandeis EM facility is supported by National Institutes of Health grant P01 GM62580.

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Name Company Catalog Number Comments
S. cerevisiae TAP tagged strain Open Biosystems YSC1177 This is the primary yeast strain used to develop the TAP protocol. Its background is S288C: ATCC 201388: MATa, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0, SNU71::TAP::HIS3MX6
Coffee grinder Mr. Coffee IDS77 Used for cell lysis
Hemocytometer, Bright Line Hausser Scientific 3120 Used to assess cell lysis
JA 9.100 centrifuge rotor  Beckman Coulter, Inc. Used to harvest the yeast cells
JA 20 fixed-angle centrifuge rotor Beckman Coulter, Inc. Used to clear the cell extract of non-soluble cellular material
Ti 60 fixed-angle centrifuge rotor Beckman Coulter, Inc. Used to further clear the soluble cell extract
Thermomixer Eppendorf R5355 Temperature controlled shaker
Novex gel system Thermo Fisher Scientific 
IgG resin GE Healthcare 17-0969-01 Sepharose 6 fast flow
Calmodulin resin Agilent Technologies, Inc. 214303 Affinity resin
Protease inhibitor cocktail, mini tablets Sigma Aldrich 589297 Mini cOmplete ultra EDTA-free tablets
Protease inhibitor cocktail, large tablets Sigma Aldrich 5892953 cOmplete ultra EDTA-free tablets
Phenylmethanesulfonyl fluoride (PMSF) Dissolved in isopropanol
2 mL Bio-spin column Bio-Rad Laboratories, Inc. 7326008 Used to pack and wash the Calmodulin resin
10 mL poly-prep column Bio-Rad Laboratories, Inc. 7311550 Used to pack and wash the IgG resin
Precast native PAGE Bis-Tris gels Life Technologies BN1002 Novex NativePAGE Bis-Tris 4-16% precast polyacrylamide gels
NativeMark protein standard Thermo Fisher Scientific LC0725 Unstained protein standard used for native PAGE. Load 7.5 μL for a silver stained gel and 5 μL for a SYPRO Ruby stained gel
Precast SDS PAGE Bis-Tris gels Life Technologies NP0321 Novex Nu-PAGE Bis-Tris 4-12% precast polyacrylamide gels 
PageRuler protein standard Thermo Fisher Scientific 26614 Unstained protein standard used for Western blotting
SDS running buffer Life Technologies NP0001 1x NuPAGE MOPS SDS Buffer
TAP antibody Thermo Fisher Scientific CAB1001 Primary antibody against CBP tag
Secondary antibody Thermo Fisher Scientific 31341 Goat anti-rabbit alkaline phosphatase conjugated
BCIP/NBT Thermo Fisher Scientific 34042 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium 
Dialaysis units Thermo Fisher Scientific 88401 Slide-A-Lyzer mini dialysis units
Centrifugal filter units, 100kDa MWCO EMD Millipore UFC5100008 Amicon Ultra-0.5 Centrifugal Filter Unit with Ultracel-100 membrane
Detergent absorbing beads Bio-Rad Laboratories, Inc. 1523920 Bio-bead SM-2 absorbants
SYBR Green II Thermo Fisher Scientific S-7564 Flourescent dye for nucleic acid staining, when detecting with SYPRO Ruby present, use excitation wavelength of 488 nm and emission wavelength of 532 nm
SYPRO Ruby Molecular Probes S-12000 Flourescent dye for protein staining, when detecting with SYBR Green II present, use excitation wavelength of 457 nm and emission wavelength of 670 nm
Copper grids Electron Microscopy Sciences G400-CP

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