Purification of Ribonucleoproteins Using Peptide-Elutable Antibodies and Other Affinity Techniques

  • Scott W. Stevens
Part of the Methods in Molecular Biology book series (MIMB, volume 488)

Summary

Recently developed affinity purification methods have revolutionized our understanding of the higher-ordered structures of multisubunit, often low-abundance macromolecular complexes, including ribonucleoproteins (RNPs). Often, purification by classical, non-affinity-based techniques subjects salt-labile complexes to an ionic strength incompatible with the integrity of the RNP, leading to a misrepresentation of the true higher-ordered structure of these complexes. A family of plasmids has been generated that can be used to introduce a number of different epitope tags, including peptide-elutable affinity tags, into the genome of the yeast Saccharomyces cerevi-siae. Alternatively, these plasmids may be used for plasmid-borne expression of epitope-tagged proteins in either yeast or Escherichia coli. The gentle elution of the complex from the antibody affinity matrix can be performed at 4 °C and is compatible with a range of salt and pH conditions. RNPs purified by this method are active and suitable for downstream analyses such as RNA sequencing, structural analysis, or mass spectrometry peptide identification.

Key Words

Affinity purification peptide antibody ribonucleoprotein 

1. Introduction

Ribonucleoproteins (RNPs) are composed of at least one molecule of RNA and one or more proteins and are ubiquitously found in all organisms. The most abundant RNP, the ribosome, has been studied extensively for many decades due to its high abundance and ease of preparation. Prior to the advent of genetically engineered epitope tags, low-to-moderate abundance RNPs had been difficult to purify from eukaryotic cells, especially yeast. Recently, high-efficiency, high-affinity epitope tags have been developed that can be engineered into the native chromosomal context of the gene of interest to reduce nonspecific effects sometimes seen with expression of proteins at nonnative levels (1, 2, 3). A system has been developed for using peptide-specific monoclonal antibodies to affinity purify peptide-tagged proteins from complex extracts alone or in conjunction with other affinity techniques to produce highly pure RNP complexes. This method involves the use of free peptide to disrupt the antibody-antigen interaction. In this chapter, details are provided for (1) epitope tagging genes in the chromosome of yeast, (2) preparation of mid- to large-scale whole-cell extracts, (3) affinity purification of the species of interest, and (4) downstream analysis of the purified material.

2. Materials

  1. 1.

    Appropriate tagging vector.

     
  2. 2.

    Monoclonal antibody of interest, protein G agarose, Ni-NTA agarose, calmodulin agarose.

     
  3. 3.

    Oligonucleotide primers for polymerase chain reaction (PCR) amplification, dNTPs (deoxynucleotide 5′-triphosphate), PCR enzymes.

     
  4. 4.

    Yeast strain of interest with desired genotype.

     
  5. 5.

    YPD liquid: 1% yeast extract (Difco), 2% Bacto Peptone (Difco), 2% dextrose.

     
  6. 6.

    Selective dropout media: 0.67% yeast nitrogen base without amino acids (Difco), required amino acid/nucleotide supplements (Bio101), 2% dextrose. (For plates, add 2% agar.)

     
  7. 7.

    Solutions used in yeast transformations: 50% PEG3350, 1 M. LiOAc, 2 mg/mL salmon sperm DNA.

     
  8. 8.

    Lyticase solution: 1 .M sorbitol, 10 mM Tris-HCl, pH 7.5, 10 mg/mL lyticase (Sigma cat. no. L4025).

     
  9. 9.

    Spheroplast wash buffer: 1 M sorbitol, 10 mM Tris-HCl, pH 7.5.

     
  10. 10.

    Lysis buffer A solution: 2 N NaOH, 8% β-mercaptoethanol.

     
  11. 11.

    Lysis buffer B solution: 50% trichloroacetic acid.

     
  12. 12.

    Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer: 100 mM Tris, pH 6.8, 2% SDS, 100 mM dithiothreitol (DTT), 15% glycerol, 0.05% bromophenol blue.

     
  13. 13.

    SDS-PAGE gels and electrophoresis apparatus, electroblot apparatus, nitrocellulose membrane.

     
  14. 14.

    Horseradish peroxidase (HRP)-conjugated antimouse immunoglobulin G (IgG) secondary antibody or rabbit PAP (Sigma P1291).

     
  15. 15.

    Protein G agarose (GE Biosciences).

     
  16. 16.

    IPP100: 10 mM Tris-HCl, pH 8.0, 100 mM NaCl (note IPP150 contains 150 mM NaCl), 0.1% NP-40.

     
  17. 17.

    0.2 .M sodium borate, pH 9.0.

     
  18. 18.

    0.2 M ethanolamine, pH 8.0.

     
  19. 19.

    Dimethylpimelimidate (DMP).

     
  20. 20.

    Phosphate-buffered saline (PBS), 1X: 10 mM Na2HPO4, 2 mM KH2PO4, 2.7 m.M KCl, 137 mM NaCl.

     
  21. 21.

    Buffer A: 10 mM HEPES, pH 8.0, 10 mM KCl.

     
  22. 22.

    Chrontrol XT timer (Chrontrol, San Diego, CA).

     
  23. 23.

    Bead-beater apparatus (BioSpec), 0.5-mm glass beads.

     
  24. 24.

    Ti-45 or Ti-60 (or equivalents) ultracentrifugation rotors and ultracentrifuge

     
  25. 25.

    Chromatography columns (0.7 × 5 cm, Bio-Rad)

     
  26. 26.

    Protease inhibitors: Phenylmethylsulfonyl fluoride (PMSF), leupeptin, pepstatin, ethylenediaminetetraacetic acid (EDTA).

     
  27. 27.

    Synthetic peptide.

     
  28. 28.

    Antibody column-stripping solution: 1 M urea, 50 mM Tris, pH 8.0.

     
  29. 29.

    Phenol/chloroform (1:1), pH > 6.7.

     
  30. 30.

    3 M NaOAc, pH 5.3.

     
  31. 31.

    100% ethanol (−20 °C).

     
  32. 32.

    80% ethanol (−20 °C).

     
  33. 33.

    100% acetone (−20 °C).

     
  34. 34.

    RNA sample buffer: 8 Murea, 10 mM Tris, pH 7.0, 0.05% bromophenol blue, 0.05% xylene cyanol.

     
  35. 35.

    Optional: SW41 or equivalent rotor and tubes for velocity sedimentation gradients.

     

3. Methods

3.1. Creation of a Yeast Strain Containing a Genomically Encoded Epitope Tag

We have created a family of vectors with which one can amplify any of several tags with two gene-specific oligonucleotides. In this section, methods for inserting a tag in the genomic locus of interest are outlined.

3.1.1. Targeting Fragment PCR Amplification and Purification

In Table 1, the various tags available in the family of vectors are shown. In addition to the peptide-antibody affinity tags, others are available that can be used alone or in conjunction with the peptide epitopes on other polypeptides in multiprotein complexes (seeNote). Oligonucleotides are designed using the format presented in Fig. 1. Essentially, the 3′ ends of the oligos are fixed (cyan and red in Fig. 1) and will anneal to the appropriate locations in all of the plasmids described in Table 1. The 5′-most 50 nt of the oligos are designed to be identical to the 50 nt upstream of the stop codon (Oligo KIA, green in Fig. 1) and 50 nt of sequence 20–30 downstream of the stop codon (Oligo KIB, orange in Fig. 1). These two primers are used in a PCR reaction of the following composition: 0.2 μM oligo KIA, 0.2 μM oligo KIB, 25 ng plasmid, 250 μM dNTPs, using a proofreading enzyme and buffer to reduce mutations resulting during PCR amplification (4). Amplification is performed as follows: 25 cycles at 94 °C for 30 s, 55 °C for 3 min, and 72 °C for 4 min. PCR products must be separated from the plasmids, which can autonomously replicate in yeast and are preparatively purified from 1% agarose gels using the Qiagen (or similar) gel purification kit.
Table 1

A Family of Oligonucleotide-Compatible Vectors for Epitope Tagging Genes

Plasmid series

Auxotrophic markers availablea

Epitope tag

Elution procedure

 

pPyxxxb

H, L, W, K

Polyoma epitope

MEYMPME peptide

 

pCT-1xxx

H, L, W

CT-1 epitope

GRILTLPRS peptide

 

pCBPxxx

H, L, W, K

Calmodulin-binding peptide

4 mM EGTAc

 

pHISxxx

H, L, W, K

Polyhistidine

200 mM imidazole

 

pCHPxxx

H, L, W, K

HIS-tag and polyoma epitope

Imidazole + Py peptide

 

pPrAxxx

H, L, W, K

Protein A (PrA)

None, used for negative selection

 

pTAPxxx

L, W, U

CBP + PrA; TEVd site

TEV cleavage; EGTA

 

aH, HIS3; L, LEU2; W, TRP1; K, KAN (G418r).

bxxx corresponds to the naming convention of the pRS series of vectors (6). cEthylene glycol-bis(2-aminoehtylether)-N,N,N ′,N ′-tetraacetic acid, pH 8.0. dobacco etch virus protease cleavage site.

cEthylene glycol-bis(2-aminoehtylether)-N,N,N′,N′-tetraacetic acid, pH 8.0.

dobacco etch virus protease cleavage site.

Fig. 1.

Design of oligonucleotides and experimental schema for epitope tag insertion into the yeast genome. (A) Schematic of the use of gene-specific oligonucleotide primers on a generic epitope tag-containing vector of the series described in the text. (B) Reaction scheme for homologous recombination of resulting polymerase chain reaction (PCR) fragment into the genome of yeast. Targeting of Your Favorite Gene (YFG) by design of the PCR primers as described is accomplished by homologous recombination directed by gene-specific sequences preceding the stop codon (green) and downstream of the stop codon (orange). Plasmid-specific sequences common to all plasmids are represented in cyan and red. Epitope tag sequences are represented in purple, and the auxotrophic marker sequences are represented in yellow. (C) Specifics of the oligonucleotide design for this family of vectors. Top: Example of oligonucleotide design for the SNU17 gene of yeast. The stop codon is in bold (TAA). Colors of the sequences correspond to the descriptions in (B). Note the polarity of the sequences, as both strands are shown. Sequence in KIA oligo is in the sense strand; sequence in KIB oligo is in antisense strand. Middle: Examples of sequences in conjunction with the plasmid-specific sequences (cyan and red). Bottom: Expected polypeptide additions to the end of the polypeptide from the targeted gene for comparison to DNA sequencing reactions (see text). (See Color Plate)

3.1.2. Transformaion of Yeast With PCR Fragment

High-efficiency yeast transformation is required to obtain sufficient numbers of homologously targeted colonies. Detailed protocols are available elsewhere (5); briefly:
  1. 1.

    The strain of interest is grown in 50–100 mL YPD to an optical density (OD) (A600) of 0.7 to 1.0. Cells must be actively growing and cannot simply be diluted to the desired density. It is preferable to use derivatives of the strain S288C, the genome of which has been sequenced (seeNote 2) (6).

     
  2. 2.

    Cells are pelleted in 50-mL conical tubes in a clinical tabletop centrifuge, washed once with sterile water, resuspended in 1 mL 100 mM LioAc, and transferred to a microcentrifuge tube that has volume markings.

     
  3. 3.

    Cells are pelleted at 5000 g for 1 min in a microcentrifuge and the supernatant removed.

     
  4. 4.

    Estimate the packed cell volume and add 1.5 volumes 100 mMLiOAc.

     
  5. 5.

    Vortex to resuspend cells, aliquot in 50-μL portions into as many tubes as you require. Two additional tubes should be included, one for a positive control, one for a negative control.

     
  6. 6.

    Tubes are centrifuged at 5000 g for 1 min and the supernatant removed.

     
  7. 7.

    To each tube, add 240μL 50% PEG3350, 36 μL 1 M LiOAc, 50 μL 2 mg/mL boiled and subsequently chilled salmon sperm DNA.

     
  8. 8.

    For a positive control, add 34 μL of 1 ng/μL of a vector that will confer growth on the medium dictated by the auxotrophic marker in the plasmid used in Subheading 3.1.1.

     
  9. 9.

    To the negative control, add 34 V03BC;L sterile water.

     
  10. 10.

    To each experimental tube, add 34 μL of the purified PCR fragment from Subheading 3.1.1.

     
  11. 11.

    Vortex all tubes well and incubate at 30 °C for 30 min.

     
  12. 12.

    Transfer to 42 °C for 45 min.

     
  13. 13.

    Pellet the cells by microcentrifugation at 5000 g for 1 min, remove supernatant, and resuspend cells in 100 μL water.

     
  14. 14.

    Plate on proper selective SD medium (7). To avoid plating too many colonies on one plate, use two plates per transformation, one with 95 μL of transformation mix and one plate with 95 μL water plus 5 μL transformation mix.

     
  15. 15.

    Incubate at 30 °C for 3–5 d, when colonies generally appear.

     

3.1.3. Confirmation of Correct Targeting and Functional Epitope Addition

There are two ways by which one can verify the proper homologously targeted epitope tag. The first is a PCR-based screen, followed by DNA sequencing (useful for all but the protein A and TAP tags). The second is to functionally test for the presence of the epitope tag in a Western blot (useful for all but the HIS and CBP tags).

3.1.3.1. Correct Targeting
To perform a PCR-based screen, one requires oligonucleotide primers designed as shown in Fig. 1. One is designed approx 200 nt upstream of the stop codon of the targeted gene (UP oligo in Fig. 1B); the other is fixed in the terminator sequence derived from the targeting plasmid (DOWN oligo [5′-CCTACAATGATGAATGCGTTTCTGCCG-3′] in Fig. 1B).
  1. 1.

    Colonies (̃4–8) resulting from the transformation in Subheading 3.1.1. are grown individually in 3 mL YPD overnight at 30 00B0;C with shaking.

     
  2. 2.

    The next morning, 0.5 mL is aseptically removed from each tube, and cells are pelleted in a microcentrifuge at 14,000 g for 1 min.

     
  3. 3.

    Cells are washed once in 1 mL water and repelleted.

     
  4. 4.

    Cells are then resusupended in 1 mL of a solution of lyticase solution.

     
  5. 5.

    ubes are incubated at 25°C to 30 °C for 30–60 min on a rotator or a nutator. This treatment will remove the yeast cell wall (seeNote 3).

     
  6. 6.

    After cells have been spheroplasted, they are gently pelleted by spinning in a microfuge at 5000 g for 1 min.

     
  7. 7.

    After removing the supernatant, they are washed once with 1 mL of spheroplast wash buffer and repelleted.

     
  8. 8.

    Remove the wash solution and add 100 μL of 1X PCR buffer (seeNote 4) and pipet up and down to mix.

     
  9. 9.

    Cells are lysed by incubation at 100 °C for 5 min.

     
  10. 10.

    Cell debris is pelleted in a microcentrifuge for 10 min at 14,000 g at 4 °C.

     
  11. 11.

    Immediately remove the supernatant and transfer into a fresh tube.

     
  12. 12.

    Use 5 μL of the supernatant in a 100-μL PCR reaction of the following composition: 0.2 μM oligo UP, 0.2 μM oligo DOWN (see Fig. 1B), 250 μM dNTPs, using a proofreading enzyme and appropriate buffer. Twenty-five cycles of amplification are performed as follows: 94 °C for 30 s, 55 °C for 60 s, and 72 °C for 1.5 min.

     
  13. 13.

    PCR products are electrophoretically separated from primers and genomic DNA on a 2% agarose gel and purified with the Qiagen gel purification kit. If UP and DOWN oligonucleotides are designed according to the scheme presented in Fig. 1, the PCR fragment should be approx 300 bp. To confirm the in-frame incorporation of the epitope tag, compare the theoretical sequence of the gene and the additional sequences contributed from the epitope tag (see Fig. 1C) against that realized from sequencing the PCR fragment with the UP or DOWN oligonucleotide.

     
3.1.3.2. Functional Analysis of Epitope Tag Incorporation by Western Blot
An alternative, or perhaps additional, means of demonstrating that the epi-tope has been properly inserted into the gene of interest is to test by Western blotting. The method of Yaffe and Schatz (8) is used to prepare whole-cell protein samples from cells for Western blot analysis, a rapid method amenable to simultaneous processing of several samples.
  1. 1.

    Briefly, 1 mL of cells grown as in Subheading 3.1.3.1. is chilled to 4 °C in ice.

     
  2. 2.

    Add 150 μL ice-cold lysis buffer A solution to each sample.

     
  3. 3.

    Mix by inverting tube several times.

     
  4. 4.

    Incubate on ice for 10 min.

     
  5. 5.

    Add 150μL ice-cold lysis buffer B; mix by inverting tube several times.

     
  6. 6.

    Incubate on ice for 10 min.

     
  7. 7.

    Pellet the precipitated proteins for 2 min at 14,000 g in a microcentrifuge.

     
  8. 8.

    Wash pellet with 1 mL ice-cold acetone.

     
  9. 9.

    Spin 2 min at 14,000 g in a microcentrifuge and aspirate the supernatant.

     
  10. 10.

    Dry pellet for 10 min uncapped on the benchtop.

     
  11. 11.

    Resuspend pellet in 100 μL SDS-PAGE sample buffer. The cell material can be very difficult to resuspend. Try adding approx 20 μL and smearing the pellet along the side of the tube with a pipet tip, then add the remaining 80 μL and mix thoroughly. If the color is yellow, quickly add 10 μL 1 MTris, pH 8.0, and vortex to mix.

     
  12. 12.

    Heat at 95 °C for 5 min.

     
  13. 13.

    Load 10 μL on an SDS-PAGE gel appropriate for re-solving your proteins of interest.

     
  14. 14.

    Electroblot the contents of the gel to a nitrocellulose membrane overnight. In our hands, wet transfer is much more efficient than a semidry transfer for this application.

     
  15. 15.

    The appropriate monoclonal antibody is used at 10–25 ng/mL in a Western blot.

     
  16. 16.

    Secondary antibodies for the Py and CT-1 epitopes can be HRP- or AP-conjugated antimouse IgG, and detection should be done with an appropriate chemiluminescent detection substrate. The presence of an appropriate size band in experimental strains, but not in a negative control, in addition to a confirmatory PCR reaction should give one confidence to proceed. Anti-HIS antibodies are not sufficiently sensitive enough to detect HIS tags on low- to medium-abundance proteins. TAP-tag and protein A detection is achieved using the rabbit PAP reagent from Sigma (P1291).

     

3.2. Preparation of Affinity Resin

Proper affinity resin preparation is critical for efficient affinity purification of RNP complexes. If the antibody is not quantitatively covalently linked to the resin, it will slowly leach off the column during purification, reducing your final yield. The polyoma antibody (9) is available commercially from Covance (GLU-GLU epitope); however, for large-scale applications, it is more economical to grow the hybridoma cells expressing the polyoma antibody. The CT-1 antibody (10) is not currently commercially available.

3.2.1. Growth of Hybridoma Cells

The hybridoma cell lines described here grow well in protein-free hybridoma media (PFHM II from Invitrogen). It is best to start from a frozen ampule of cells and slowly grow to large volumes. A cryopreserved ampule of the hybridoma cells is thawed and added to 10 mL of PFHM II in a T-25 flask and grown until confluent. Growth is performed in a humidified CO2 incubator at 37 °C. From there, the culture is expanded into two T-75 flasks. On reaching confluence in those flasks, expand into the necessary number of T-175 flasks and grow until cells justbegin to die. Another measurement of when the supernatant is ready to be processed is analysis of protein concentration in the cell-free culture supernatant. Since the PFHM II is protein free, when the concentration of antibodies secreted into the medium reaches 50–100 ng/μL, it is ready to harvest.

3.2.2. Purification of Antibodies From Hybridoma Supernatant

  1. 1.

    Centrifuge the cell culture material from Subheading 3.2.1. at 10,000 g at 4 °C for 20 min to pellet the cell debris.

     
  2. 2.

    Carefully remove the supernatant and place into a glass beaker slightly more than wice as large as the volume of supernatant at 4 °C.

     
  3. 3.

    With gentle stirring, slowlyadd an equal volume of cold, saturated ammonium sulfate solution.

     
  4. 4.

    Cover with plastic wrap and stir overnight at 4 °C.

     
  5. 5.

    Centrifuge the ammonium sulfate precipitation at top speed in appropriate size centrifuge tubes. With less than 300 mL of ammonium sulfate precipitate, spin in an SS34 (or equivalent) rotor at more than 30,000 g for 30 min. If you have more supernatant than an SS34 can accommodate, the precipitate can be centrifuged in a GSA or GS3 rotor (or their equivalents) at the maximum rated speed for the rotor for 2 h at 4 °C.

     
  6. 6.

    Carefully remove the supernatant and rinse the pellets with 50% ammonium sul-fate (seeNote 5).

     
  7. 7.

    Resuspend the precipitated material in 0.02 volume (original cell culture volume) 25 mMsodium phosphate, pH 7.0.

     
  8. 8.

    Dialyze the antibody three times against more than 500 volumes 25 mMsodium phosphate at pH 7.0 for 4 h each for the first two dialyses, with the last dialysis proceeding overnight.

     
  9. 9.

    Measure the concentration of the antibody with the Bradford assay, using IgG as a standard (11), and assess the purity of the antibody by SDS-PAGE analysis with and without DTT in the SDS sample buffer (seeNote 6). Antibodies purified by these means are generally more than 95% pure. Freeze at −20 °C to −80 °C in convenient aliquots (e.g., 20 mg/tube).

     

3.2.3. Coupling of Antibody to Agarose Beads

Orienting the antibody such that the antigen-binding domain is exposed to the solution is preferable to random coupling to the beads via cyanogen-bromide coupling, which will orient the antibodies randomly with respect to the antigen-binding domain. To perform this, use protein G agarose to bind the Fc region of the antibody, thereby exposing the Fab region to the solution.
  1. 1.

    Wash 5 mL protein G slurry twice with 45 mL IPP100 in 50-mL conical tubes.

     
  2. 2.

    The protein G agarose beads are recovered by centrifugation at approx 1000 g for 2 min in a clinical centrifuge at room temperature.

     
  3. 3.

    Resuspend the washed beads in antibody solution containing 1.5 times the manufacturer's capacity estimation for protein G binding.

     
  4. 4.

    This slurry is incubated with slow rotation at room temperature for 2 h.

     
  5. 5.

    After antibody binding, the slurry is centrifuged at about 1000 g for 2 min in a clinical centrifuge at room temperature (seeNote 7).

     
  6. 6.

    Carefully remove the antibody-depleted supernatant, which can be saved and used for Western blotting in the future.

     
  7. 7.

    Wash the beads three times with 40 mL 0.2 M sodium borate, pH 9.0, carefully removing the supernatant after each spin.

     
  8. 8.

    Resuspend the beads in 40 mL 0.2 M sodium borate, pH 9.0.

     
  9. 9.

    Add 0.233 g DMP solid to this solution to crosslink the antibody to the protein G.

     
  10. 10.

    Incubate with rotation for 30 min at room temperature.

     
  11. 11.

    Centrifuge the tube at approx 1000 g for 2 min in a clinical centrifuge at room temperature.

     
  12. 12.

    Carefully remove supernatant and resuspend beads in 40 mL 0.2 M ethanolamine, pH 8.0, to quench the crosslinking agent.

     
  13. 13.

    Incubate with gentle rotation for 2 h at room temperature.

     
  14. 14.

    Centrifuge the tube at approx 1000 g for 2 min in a clinical centrifuge at room temperature.

     
  15. 15.

    After careful removal of supernatant, wash beads three times with 40 mL 1X PBS.

     
  16. 16.

    Resuspend in 1 bead volume of 1X PBS plus 0.02% NaN3 (seeNote 8) and store at 4 %C in a tightly capped container. The efficiency of antibody crosslinking can be monitored by removing samples at each step and at the end by boiling 20 μL of beads in SDS-PAGE sample buffer and looking for the heavy chain (̃50 kDa) and light chain (̃25 kDa) in an SDS-PAGE gel.

     

3.3. Preparation of Yeast Whole-Cell Extracts

There are a number of ways to prepare large-scale yeast whole-cell extracts. Among them are (1) blending in liquid nitrogen (12,13), (2) continuous-flow high-pressure disruption, and (3) “bead beating” using glass beads in a specialized apparatus (BioSpec). In our experience, the first method is highly variable in breakage efficiency. The second method requires the acquisition of relatively expensive equipment but is a highly efficient means of processing kilogram quantities of yeast (Microfluidics). Method 3 is amenable to quantities from 80 to 500 g of yeast, generally the amount of cells used for preparative RNP purification. In this chapter, experimental details for the bead-beating procedure are provided.

3.3.1. Growth and Harvesting of Cells

If the epitope tag is incorporated in the genome, there is no further need for selection in minimal medium.
  1. 1.

    Grow cells in YPD to mid-log cell concentrations (OD A600 ̃ 1.5–2.0) in shake flasks at 30 °C or in fermenters to an empirically determined mid-log phase, which can reach optical densities of 10 or greater.

     
  2. 2.

    Cells are harvested by centrifugation at 8000 g for 10 min at 4 °C.

     
  3. 3.

    After removal of media, cell mass is determined, and cells are resuspended in 1.5 mL ice-cold buffer A per gram of cells. The low-salt conditions allow for optimization of extract procedures (seeSubheading 3.3.2.).

     
  4. 4.

    Cells are preserved by slow pipetting into a vat of liquid nitrogen, which also aids in the breakage and allows for storage at −80 °C in a convenient form.

     

3.3.2. Homogenization of Cells

  1. 1.

    The required mass of cryopreserved cells is removed from the freezer and transferred to an appropriate size glass beaker containing a stir bar (seeNote 9).

     
  2. 2.

    The beaker containing the cells is set in a room temperature water bath and occasionally stirred with a clean spatula to scrape thawing material off the sides.

     
  3. 3.

    When the material has liquefied to a custard-like consistency, but before it has completely thawed, begin stirring with the stir bar to accelerate the thawing process.

     
  4. 4.

    Monitor this carefully, and when all of the frozen material has melted, transfer the material to a cold room.

     
  5. 5.

    With stirring, add PMSF to 0.4 mM, leupeptin and pepstatin each to 2 μg/mL, and EDTA to 0.5 mM.

     
  6. 6.

    Add 200 mL of cell suspension (80 g cell mass) to the required numbers of stainless steel bead-beater chambers.

     
  7. 7.

    Fill to the manufacturer‧s recommended level with clean, cold, 0.5-mm glass beads.

     
  8. 8.

    Assemble the chamber into the ice jacket and fill ice jacket with ice water (seeNote 10).

     
  9. 9.

    Cycle 1 min on and 2 min off for five cycles. A Chrontrol XT timer (or equivalent) is very useful for automating the cycling.

     
  10. 10.

    After the cycling is complete, the contents of the chamber (including the beads) are transferred to a 1-L centrifuge bottle (or other 1-L capped container).

     
  11. 11.

    The supernatant is transferred by pipet to a cold glass beaker containing a stir bar.

     
  12. 12.

    Buffer A10 containing PMSF, leupeptin, pepstatin, and EDTA as described above is added (100 mL/chamber) to rinse the chamber and is added to the beads.

     
  13. 13.

    The bottle is capped and agitated gently to rinse the beads.

     
  14. 14.

    After the beads settle, the supernatant is transferred to the glass beaker.

     
  15. 15.

    Repeat the rinse with an additional 100 mL A10 containing protease inhibitors.

     

3.3.3. Preparation of Cell Extracts

Depending on the characteristics of the protein/RNP you are attempting to extract, you will need to add extraction agents, such as KCl, NaCl, detergents, or other salts to efficiently extract the complex of interest.
  1. 1.

    Typically, KCl is added to 200 mM final concentration, slowly, with stirring from a 2 M KCl stock.

     
  2. 2.

    After stirring for 30 min at 4 °C, cell debris is pelleted in an ultracentrifuge spun at 100,000 g for 60 min at 4 °C.

     
  3. 3.

    Soluble material from the center of the tubes is harvested, avoiding the pellet and the fatty material at the top of the tube (seeNote 11).

     
  4. 4.

    If the chromatography is going to be performed that day, this material can be filtered through a 0.45-μMfilter and applied directly to the column (seeSubheading 3.4.1.). If it is to be preserved or if other adjustments to the solution are warranted, dialysis into an appropriate solution should be performed (seeNote 12).

     

3.4. Affinity Chromatography

The affinity chromatography protocols will differ slightly depending on the affinity matrix used. Generally, antibody affinity matrices require slow passage of the extracts over the material. Affinity matrices with much greater affinity (such as Ni-NTA) are used with greater flow rates.

3.4.1. Preparation of Affinity Column

The antibody affinity column is assembled using the antibody-agarose conjugate prepared in Subheading 3.2.3.b An empirically determined amount of antibody slurry is loaded into a small column and allowed to drain (seeNote 13). If the abundance of the material is unknown, or if the material is of low abundance, use 0.5 mL of settled antibody bead volume per 100 g of cells. This provides approx 2–3 mg of antibody on the column under ideal conditions and will capture more than 80% of the desired material with proper running, washing, and elution conditions (2).

3.4.2. Passage of Extracts Over Affinity Matrix

Passage of extracts over the antibody affinity matrix is best done with a slow-pumping peristaltic pump with a flow rate of 5–30 mL /h. The actual flow rate is dictated by a number of variables, such as the volume of extract, a convenient length of time (overnight usually), and the strength of the antibody/antigen interaction. Using the peristaltic pump ensures that the rate remains constant; however, careful calculation must be made to ensure that the column is not pumped dry. When the last of the extract is about to be pumped through the affinity column, disconnect the peristaltic pump and drain the remains (usually ̃5 mL) into the column to flow by gravity. Alternatively, the column can be run by gravity by adjusting the height of the extract over the column to properly adjust the flow rate.

3.4.3. Washing Affinity Matrix

After all of the cell extract has passed over the column, rinse the sides of the column with wash buffer and allow to drain by gravity. Wash the column with a buffer suitable for the stability of the complex of interest. Typically, the polyoma and CT-1 antibodies are stable to washing with 100–150 column volumes of wash solution of up to 250 mM monovalent salt concentration. If the complexes are sensitive to 250 mM salt concentrations, the wash buffer salt concentration can be reduced to as low as 50 mM; however, a second affinity step will be required to reduce background binding to the agarose-based affinity matrix.

3.4.4. Affinity Elution of Bound Material

One of the primary advantages of a peptide-elutable affinity matrix is the gentle elution procedure. The free peptide can be easily removed by ultrafiltration, size-exclusion chromatography, or sedimentation through velocity gradients (seebelow). Other affinity matrices sometimes require the use of proteases, which can be difficult to remove after use, or divalent metal ion chelators such as EGTA (ethylene glycol tetraacetic acid), which in some cases can remove structural metals from RNPs, affecting their integrity (unpublished observations). Use 100 ng/mL peptide dissolved in wash buffer (seeNote 14). The competitive elution procedure produces highly concentrated purified material if the flow is interrupted during the elution step. Apply the elution buffer to the top of the column and allow one column volume to drain by gravity. Flow is stopped for 30 min. This is repeated for five cycles to remove as much of the material as possible. The majority of affinity-purified material is generally contained in the second and third fractions. Antibody columns can be regenerated by washing with 20 column volumes of antibody column stripping solution, then by washing with IPP150 containing 0.02% NaN3. Columns may be stored for over a year at 4 °C.

3.4.5. (Optional) Second Affinity Chromatography Steps

Often, a second affinity step can increase the purity of the complexes of interest (1,2). In multicomponent complexes, this can be achieved by incorporation of a second affinity tag from Table 1 into the same gene as the peptide epitope (e.g., CHP tag, which incorporates a polyhistidine sequence in tandem with a polyoma epitope) or by incorporating a second affinity tag into a separate polypeptide (e.g., polyhistidine into one polypeptide and polyoma into a different polypeptide present in the same complex). Note that Ni-NTA should not be used as a first purification step from yeast extracts. At low-salt concentrations, antibody affinity columns are efficient at capturing the epitope-tagged polypeptide; however, the binding of other, nonspecific material is higher. An example of low-salt-purified SmD3-CHP-associated RNA is shown in Fig. 2. Material purified only through polyoma chromatography is presented in Fig. 2A, and equal amounts of material further purified by Ni-NTA chromatogra-phy is shown in Fig. 2B. Ribosomal RNA, which is often the most abundant contaminant in these procedures, is undetectable after Ni-NTA purification when purification is performed at 50 mM KCl. Additional tags available are an additional peptide-elutable antibody epitope, polyhistidine, calmodulin-binding peptide, protein A, and TAP (1). As others have shown, protein A can be used to negatively select against related, but unwanted, complexes that happen to share a polypeptide, an additional utility that has proven very useful (14). Note that protein A-containing polypeptides will also be captured in the Py and CT-1 columns but not eluted by the peptide. Proper experimental design is required to make use of these tags in conjunction with the peptide-elutable affinity tags.
Fig. 2.

Resulting RNAs affinity purified from a strain harboring a CHP tag on the SmD3 polypeptide and stained with silver. (A) SmD3CHP-associated RNA purified in a single step over an antipolyoma affinity matrix as described in the text. Note the contaminating 25S, 18S, 5.8S, and 5S ribosomal RNAs (rRNAs) in addition to the U1, U2, U4, U5, and U6 small nuclear RNAs. (B) Material from the polyoma purification in (A) further purified over an Ni-NTA affinity matrix. Note the absence of the rRNAs after the second affinity step.

3.5. Downstream Analysis of Affinity-Purified Material

Even if the material purified by these means were 100% pure, the story is often more complex than is observed by SDS-PAGE analysis of the copurifying polypeptides. This is due to (1) the potential for an extramolar presence of the tagged polypeptide; (2) presence of multiple different, albeit related, complexes in which the tagged polypeptide functions (e.g., the U5 and U4/U6∙U5 snRNPs (small nuclear RNPs); 15, 16, 17); and (3) potentially substrate-engaged RNPs, which will likely be larger and more compositionally complex. To adequately address these issues, physical separation must be achieved, generally by glyc-erol gradient sedimentation or by size-exclusion chromatography. From there, polypeptides and RNA can be characterized.

3.5.1. Separation of Complexes Based on Size

A tagged polypeptide will often be a component of several different complexes. An example of this is the Sm complex, which binds to each of the spliceosomal U1, U2, U4, and U5 snRNAs. Although functionally related, the U1, U2, and U4/U6∙U5 snRNPs are separate entities at high-salt concentrations. To separate complexes based on their molecular weight, one can use size-exclusion chromatography or glycerol gradient sedimentation to separate them. The specifics of the separation techniques are particular to the size of the complex present in the purified material. In our experience, size-exclusion chroma-tography techniques result in the loss of significant quantities of material, and for precious low-abundance samples may not be the best means of separation. For RNP separation, glycerol gradient sedimentation is preferred because it does not suffer from the losses encountered on gel filtration columns.

In Table 2, the conditions for separation of different size complexes are presented. Typically, sedimentation is performed at 4 °C at the indicated speeds and times. All information is based on an 11-mL gradient used in a Beckman SW41 rotor configuration, although smaller tubes and different rotors can be used. Gradients are harvested either from the top with a manual pipetting technique or through the bottom or the top using a gradient fractionator. If there is appreciable precipitated or otherwise pelleted material at the bottom of the tube, harvesting from the bottom leads to contamination of every fraction. Fractions of 420 μL each are harvested from the top of the gradient, which provides good resolution of the resulting material.
Table 2

Sedimentation Conditions for Glycerol Velocity Gradientsa

Size of complex

Glycerol range (%)

Speed (rpm)

Time

 

<100kDa

5–20

36,000

24 h

 

100–1000kDa

10–20

29,000

18 h

 

250–3000kDa

10–30

29,000

18 h

 

500–6000kDa

10–40

22,000

16 h

 

40Sb–300S

10–50

40,000

100 min

 

a.Values given for an 11-ml gradient in SW40 or SW41 rotor or its equivalent at 4 °C.

bSvedberg constant.

RNA and protein from these fractions can be separated by extracting with an equal volume of phenol:CHCl3 (1:1), pH > 6.7. This extraction should be performed by vortexing thoroughly (1–2 min) and centrifugation at 14,000 g for 10 min at room temperature (seeNote 15). Pipet the aqueous (top) layer into a separate tube containing 42 μL 3 MNaOAc, pH 5.3. Vortex and add 1 mL ice-cold 100% ethanol. Vortex to mix and store at −20 °C overnight. To the organic phase, fill the rest of the tube with acetone (̃1.2 mL) that has been prechilled to below −20 °C. Layer the acetone carefully on the organic phase and place the tubes at −20 °C overnight without mixing. Incubation in this manner results in more efficient precipitation than mixing prior to placing at −20°C.

The next morning, invert all tubes several times and centrifuge both the RNA samples (ethanol precipitations) and the protein samples (acetone precipitations) at 4 °C for 15 min. Wash each pellet carefully with 80% ethanol (−20 °C) and let air dry for 10–20 min. RNA samples are resuspended in RNA sample buffer and are electrophoresed on an appropriately configured urea-PAGE gel (configuration will depend on the size of the RNA to be separated), and protein samples are resuspended in SDS-PAGE sample buffer and electrophoresed on an appropriately configured SDS-PAGE gel. Stain each gel according to the abundance of the RNA and proteins you are expecting, use ethidium bromide staining for abundant RNA (18), Coomassie blue G250 staining for abundant protein (19), and silver staining for low-abundance RNA and proteins (20).

3.5.2. Mass Spectrometric Analysis of Copurifying Polypeptides

Mass spectrometric peptide identification of complex mixtures is a facile and robust means of identifying copurifying polypeptides (21, 22, 23, 24). The services are commercially available, and many institutions have the means to identify proteins in your samples directly from complex mixtures or from isolated poly-acrylamide gel bands. Although the techniques for performing these analyses are beyond the scope of this chapter, here are some helpful suggestions for submitting samples that will improve the chances of obtaining good data from the mass spectrometric analysis. First, cleanliness is crucial to avoid contaminating your sample with keratin and other human polypeptides, which can mask the identity of the unknown polypeptides in your samples. Investigators working with RNA typically employ laboratory techniques to avoid RNases, which will also minimize the introduction of human contaminants. The way in which the gels are stained can have an impact on acquisition of interpretable mass spectra. The means and specifics of gel staining can also influence the results of mass spectrometry. Colloidal Coomassie staining of gels (19) and a particular silver staining technique (20) will improve the chances of success. Also, preservation of gel slices in a relatively dry state and freezing at −80 °C are recommended.

3.6. Expression of Epitope-Tagged Genes From Plasmids in Escherichia coli or Saccharomyces cerevisiae

Although the expression of genes in yeast from their natural promoter is an optimal and more representative means of studying multisubunit complexes, there may be times when that is not the best way to introduce an epitope-tagged protein into the system under investigation. The vectors described in Table 1 can be used to drive plasmid-borne epitope-tagged genes in yeast and E. coli. For designing yeast constructs, PCR can be performed from yeast genomic DNA, which will include the native promoter, and cloned in-frame with the epitope tags. Complementary DNAs from other organisms can be cloned in-frame to the epitope tags but require the addition of a yeast promoter to drive expression. Choice of plasmid copy number will allow low or high levels of expression depending on the needs of the experiment. Expression in E. coli can be achieved by cloning the desired gene in-frame with respect to the epitope tag and including an appropriate promoter and ribosome-binding site in the primer design at the 5′ end of the gene. This may be most easily achieved using the T7 RNA polymerase system (25).

3.7. Summary

The use of peptide-elutable epitope tags alone or in combination with other affinity techniques provides a gentle and efficient means of purification of even low-abundance material from yeast or E. coli cell extracts. The family of vectors presented here provides great flexibility with respect to auxotrophic markers and tags using a single pair of oligonucleotides per gene.

4. Notes

  1. 1.

    The family of vectors includes both peptide epitopes alone and in conjunction with a polyhistidine epitope tag. Should the genotype of the strain in use allow, multiple tags can be place on multiple genes, or in situations designed to test the higher-ordered organization of a polypeptide (monomer vs dimer, etc.), one can tag each allele in a diploid with a different tag.

     
  2. 2.

    Using S288C derivatives has several advantages; the sequenced genome is on occasion slightly different from other isolates, such as W303. These small differences can have downstream consequences in strain construction and peptide analysis. Among some of the advantages to using the sequenced strain are (1) increased frequency of homologous recombination in a gene for which there is sequence heterogeneity at the locus of interest, (2) confidence in the copy number of the gene, and (3) comprehensive peptide analysis on mass spectrometric pep-tide identification.

     
  3. 3.

    Spheroplasts are rather fragile, and the progress of the treatment should be carefully monitored. After spheroplasting, the cells will clump together, resulting in a stringy appearance. If the reaction is allowed to proceed too far, or if the concentration of lyticase is too high, the yield of spheroplasts will decrease due to cell lysis.

     
  4. 4.

    The composition of the PCR buffer will be dictated by the enzyme choice.

     
  5. 5.

    Allow the 50% ammonium sulfate wash solution to perfuse the pellet for approx 10 min before respinning at the same speed for 10 min. If the pellet is “loose” and slides down the side of the tube, it can be transferred to an SS34 tube (should the volume be large enough to have warranted the use of larger buckets for the precipitation) and spun at a higher RCF.

     
  6. 6.

    In the presence of SDS, DTT, and heat, the antibody will dissociate into the heavy (̃50-kDa) and light (̃25-kDa) chains. In the absence of DTT, the antibody will migrate in the approx 150-kDa range. The composition of the SDS-PAGE running and sample buffers depends on the electrophoresis method used in the laboratory.

     
  7. 7.

    Centrifugation is not the ideal means of harvesting agarose beads as there is a 3–5% loss of beads in every spin. Alternatively, to increase the yield of harvested beads, one may filter at these steps using a chromatography column or a Buchner funnel with fritted disk for the washing and quenching steps.

     
  8. 8.

    Sodium azide (NaN3) is highly toxic. Handle with care.

     
  9. 9.

    If the frozen cells are slowly extruded into the liquid nitrogen, the frozen pellets have approx twice the volume of the thawed cell suspension. This approximation can be used to appropriately apportion the required amount of cells to avoid waste or the need to refreeze cells.

     
  10. 10.

    Some protocols call for the use of a salt water-ice bath. This often causes freezing of the cell/bead mixture and reduces the efficiency of cell breakage.

     
  11. 11.

    Typically, one can safely harvest approx 15 mL of extract from a Ti-60 size tube and approx 40 mL from a Ti-45 size tube. If the material is to be used to recover a particular biochemical activity, the extract may need to be harvested from a certain location in the centrifuge tube. Empirical determination of the location of the activity of interest may be required.

     
  12. 12.

    To cryopreserve the extract, dialysis into an appropriately buffered solution containing at least 20% glycerol is recommended. Generally, dialysis is performed twice for 4 h each into more than 100 extract volumes of buffer at 4 °C with stirring . Equilibration is more rapid and thorough if the dialysis bags are inverted regularly.

     
  13. 13.

    Bio-Rad Econo-columns 0.5 × 5 cm are generally used.

     
  14. 14.

    Peptides can be used from the crude synthesis preps (>80% purity) or can be purified through a SEP-PAK C-18 cartridge according to the manufacturer's instructions (to >95% purity). As the peptide is in such high molar abundance under these conditions, as long as the peptide is free from nucleases and proteases and does not affect the pH of the solution, no functional differences are noted using highly purified peptides.

     
  15. 15.

    Centrifugation at low (4 °C) temperatures results in clouding of the layers in many cases and is not recommended. Also, use of RNA phenol (pH < 6.5) is not recommended for glycerol gradient extraction as the density of high concentrations of glycerol does not allow for phase separation at this step.

     

Notes

Acknowledgments

I gratefully acknowledge the support of John Abelson, in whose laboratory these techniques were developed. I thank Christine Guthrie and Amy Kistler for suggesting the polyoma epitope and for the contribution of the hybridoma cells. This work is supported by a grant from the Welch Foundation (F-1564), the National Science Foundation (MCB-0448556), and the American Cancer Society (RSG-05-137-01-MCB).

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Copyright information

© Humana Press, a part of Springer Science + Business Media, LLC 2008

Authors and Affiliations

  • Scott W. Stevens
    • 1
  1. 1.Section in Molecular Genetics and Microbiology, Institute for Cellular and Molecular BiologyUniversity of Texas at AustinAustinUSA

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