Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets to treat fungal infections. Here we present a protocol for the specific enrichment of TAP-tagged RpdA combined with an HDAC activity assay that allows in vitro efficacy testing of histone deacetylase inhibitors.
Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genome mining of fungal secondary metabolites. Inhibitor screening, however, requires purified enzyme activities. Since class 1 deacetylases exert their function as multiprotein complexes, they are usually not active when expressed as single polypeptides in bacteria. Therefore, endogenous complexes need to be isolated, which, when conventional techniques like ion exchange and size exclusion chromatography are applied, is laborious and time consuming. Tandem affinity purification has been developed as a tool to enrich multiprotein complexes from cells and thus turned out to be ideal for the isolation of endogenous enzymes. Here we provide a detailed protocol for the single-step enrichment of active RpdA complexes via the first purification step of C-terminally TAP-tagged RpdA from Aspergillus nidulans. The purified complexes may then be used for the subsequent inhibitor screening applying a deacetylase assay. The protein enrichment together with the enzymatic activity assay can be completed within two days.
Histone deacetylases (HDACs) are Zn2+-dependent hydrolytic enzymes capable of removing acetyl groups from lysine residues of histones and other proteins. Based on the sequence similarity, HDACs are grouped into several classes1. Recently, the fungal class 1 HDAC RpdA, an ortholog of baker's yeast (Saccharomyces cerevisiae) Rpd3p, was shown to be essential for the opportunistic fungal pathogen Aspergillus fumigatus2. Therefore, RpdA has gained importance as a potential target to treat fungal infections2. Assessment of deacetylase activity in vitro is important for the characterization of enzymatic properties and allows to determine the efficacy of novel substances for inhibitor development. Although recombinant expression of a codon-optimized version of human HDAC1 in Escherichia coli has been reported recently3, attempts to express full-length RpdA in this host failed4. Furthermore, since fungal class 1 HDACs such as RpdA and HosA exert their function as multiprotein complexes, it is favorable to use native endogenous complexes for enzymatic inhibitor studies. However, due to inhibiting factors and the presence of different HDACs in fungal lysates, catalytic activity measured in whole protein extracts is relatively low and cannot be assigned to individual enzymes. Moreover, previous studies in the filamentous fungus A. nidulans identified a class 2 HDAC, HdaA, as predominant deacetylase in chromatographic fractions of fungal extracts. Thus, multiple conventional chromatographic purification steps are needed to separate non-HdaA activity from fungal strains4. The introduction of the tandem affinity purification (TAP) strategy in A. nidulans5 has significantly eased the enrichment of specific deacetylase activities. The original TAP tag is composed of two protein A domains and a calmodulin binding peptide (CBP) separated by a tobacco etch virus protease (TEV) cleavage site6. This allows for native purification and elution of tagged proteins including their interaction partners7. When using the enriched proteins for activity assays, the mild elution under native conditions by protease cleavage is an important feature of the TAP tag purification. A GFP-tagged protein, for example, can be enriched by immobilized antibodies as well, however, cannot be eluted under native conditions.
Here we provide a detailed protocol for the single-step enrichment of active RpdA complexes via the first purification step of C-terminally TAP-tagged RpdA from A. nidulans (IgG separation and TEV cleavage) for subsequent inhibitor screening applying a histone deacetylase assay. As proved to be sufficient, affinity enrichment was restricted to just one purification step also because the enzymatic activity was significantly reduced after two-step TAP purification when compared to IgG purification alone.
Nevertheless, the introduced protocol should as well be applicable for the enrichment of other tagged enzymes involved in chromatin regulation such as acetyltransferases, methyltransferases, and demethylases. By appending the second purification step of the TAP protocol, proteins co-purified with the tagged baits can be considered as complex partners of (novel) enzymatic complexes.
1. Cultivation of A. nidulans
2. Single-step enrichment of TAP-tagged HDAC (adapted from Bayram et al. 2012)12
3. Analysis of purification by SDS-PAGE and western blotting
4. Deacetylase assay using in vitro [3H] acetate-labeled chicken reticulocyte histones (adapted from Trojer et al. 2003)4
A typical outcome of the presented single-step enrichment of RpdA is shown in Figure 1 (referred to as "IgG"). For the sake of completeness, we also have included flow-through and elution fractions ("CFT" and "CE") illustrating the second purification step by a calmodulin resin ("CaM") as described12. The displayed silver-stained gel (A) clearly illustrates the efficacy of the first affinity step that is even further increased when performing the tandem purification. Most prominent proteins present in the protein extract and the flow-through, however, already are depleted in the TEV eluate (TE). It is important to notice that the TEV elution fractions are >100 × concentrated compared to extract and flow-through. The asterisks mark tagged RpdA (compare panel B). The calculated MW of RpdA including the CBP (RpdA::CBP) is 82 kDa, however, the protein migrates at a much higher apparent molecular weight of approx. 120 kDa. This phenomenon has been observed previously and can be assigned to the specific properties of its C-terminus4,17. The immunoblot (B) shows strong signals migrating at approx. 120 kDa corresponding to CBP-tagged full-length RpdA (RpdA::CBP) in the TEV eluate ("TE"), the calmodulin flow-through ("CFT"), and eluate ("CE") fractions. In the acid eluate ("AE") a second signal with a slightly larger MW is visible. This represents the proportion of TAP-tagged RpdA bound to the IgG resin that was not released by TEV cleavage. The difference in size corresponds to 16 kDa of the protein A repeat of uncleaved RpdA::TAP. As expected, no bands could be detected by the anti-CBP antibody in the wild-type control fractions (panel B, "wild type").
The results of a representative deacetylase activity assay with the specific HDAC inhibitor trichostatin A (TSA) are displayed in Figure 2. This assay was originally developed for plants18 and has also been used for inhibitor screening against mammalian deacetylases19,20. The histones used for the assay were labeled and prepared as described16. The effect of increasing concentrations of TSA on the catalytic activity of the enriched RpdA complex ("TE ± TSA") is shown. The sensitivity of the activity confirms that measured cpm values are indeed due to RpdA and not caused by unspecific protease activity. This is an important observation as it indicates that TEV, which is present at rather high concentration, does not interfere with the HDAC activity assay. In order to assign measured HDAC activity to RpdA, a wild-type strain was used as negative control ("Co"). As expected, only marginal HDAC activity (approx. 5-10% of RpdA-enriched fractions) was detected in the TEV eluate of the wild type. Interestingly, HDAC activity is significantly reduced after the second affinity purification step ("CE") when compared to the TEV eluate.
Figure 1. Tandem Affinity purificationof TAP-tagged RpdA. A silver-stained 10% SDS-polyacrylamide gel (A) and a western blot probed with the anti-CBP antibody (B) are displayed. Lane labeling and loaded volumes are as follows: "M": 2 µL of 1:10 diluted unstained protein marker (silver stain), 3.5 µL of prestained protein marker (western blot); "Ex": protein extract sample as prepared in step 2.2.8 (2 µL of 1:10 dilution, 5 µL); "FT": IgG resin flow-through sample as prepared in step 2.4.3 (2 µL of 1:10 dilution, 5 µL); "AE": acid eluate of step 2.4.14 (10 µL, 10 µL); "TE": TEV eluate of overnight elution by TEV cleavage, step 2.4.12 (10 µL, 10 µL); "CFT": calmodulin flow-through (20 µL, 20 µL); "CE": calmodulin eluate (10 µL, 10 µL). Size of selected marker proteins is indicated on the left side of the panels. The volumes given in parentheses correspond to sample loadings for silver stain and western blot, respectively. Asterisks in the silver-stained gel indicate the RpdA fusion protein. The immunoblot (B) was detected with alkaline phosphatase using the BCIP/NBT color development system. Please click here to view a larger version of this figure.
Figure 2. HDAC activity assay under increasing concentrations of trichostatin A. Efficacy of RpdA inhibition was tested with 25 µL of affinity-purified recombinant RpdA ("TE") and 25 µL of 0, 10, 50, and 500 nM of the HDAC inhibitor TSA diluted in RPMI-1640 medium. RPMI was used to assess background activity ("blank"). Activities of the final eluate after the second calmodulin affinity step ("CE") and of an untagged strain after the first affinity purification step (negative control, "Co") are displayed. Activities are shown as percent of enriched RpdA without TSA (100 %, "TE"). Error bars indicate the standard deviation of three replicates. This figure has been modified from Bauer et al. 20162. Please click here to view a larger version of this figure.
This protocol describes a single-step enrichment of a TAP-tagged class 1 HDAC from the filamentous fungus A. nidulans for the assessment of in vitro deacetylase activity. The TAP tag was originally introduced in baker's yeast for the identification of protein-protein interaction partners of the tagged protein6. Subsequently, the tag was codon-optimized for its use in A. nidulans5. Here we provide a straight-forward step-by-step protocol for the application of the first affinity purification step of the TAP strategy for single-step enrichment of the class 1 HDAC RpdA. The second affinity purification step clearly increases the level of purification, which is particularly important for the identification of bait-interacting proteins. Nevertheless, just the first step is recommended for subsequent activity testing, since the lack of significant contamination after the first step was confirmed by a control experiment using a wild-type strain. Furthermore, eluted activity is considerably higher after single-step enrichment when compared to that of the full TAP. In addition to RpdA2, this protocol was also successfully used for purifying A. nidulans complexes of the second class 1 HDAC HosA21.
Due to our observations that fungal class 1 HDACs build up stable complexes4, we have succeeded to modify the protocol by Bayram et al. 201212 that represents the basis of this method. Nevertheless, some critical steps have to be mentioned. The preparation of highly concentrated protein extracts to ensure near-physiological conditions is critical for complex stability. Therefore, it is important to mind the given recommendations regarding the biomass/extraction buffer ratio. In this respect, it is also critical to use well ground fine mycelial powders to ensure proper extraction. Here, the use of a grinding machine is clearly advantageous. As mentioned in the protocol section, it is worth to try the TEV-cleavage step for 1-2 h at room temperature in order to speed up the purification. This was tested for RpdA without observing any deleterious effects on stability (unpublished observation, Bauer I, 2018). In addition, replacement of NP40 (used in the original protocol) with TX-100 might not be suitable for other protein complexes. When using this method for purification of other TAP-tagged proteins, one should also refer to the Bayram protocol, which contains a number of valuable hints that might be helpful for a sufficient purification of other protein complexes12.
Besides the here described TAP-method, other affinity tags and techniques are commonly used for single-step enrichment of protein of filamentous fungi, including His- and GFP-tags. However, as class 1 HDACs generally are functional as high-molecular-weight complexes, native elution conditions are a prerequisite for the enrichment of catalytically active HDACs. Importantly, many other affinity purifications are performed under unfavorable conditions. For instance, enrichment of HDACs via GFP-trap, which is based on antigen-antibody interaction, is not suitable due to the acidic elution conditions interfering with protein-protein interaction of HDAC complexes bound to resins. Moreover, attempts to purify His-tagged RpdA by metal chelate affinity chromatography22, resulted in a significant loss of catalytic activity during the purification procedure although imidazole instead of low-pH conditions was used for elution (unpublished data, Bauer, I, 2010).
One limitation of the described enzymatic assay protocol is the use of the radioactive substrate. However, assays on a fluorescent basis have been developed as well23,24 and are commercially available. These assays are performed in well-plates and thus are suitable also for high-throughput screening of HDAC inhibitors. In that case, an upscale of the presented procedure would be required.
Potential upcoming applications of this protocol include the enrichment of specific sub-complexes of class 1 HDACs to assess their specific physiological roles and/or differences in their susceptibility to HDAC inhibitors. When establishing the described method for other enzymes, it is strongly recommended to perform a negative control experiment with an untagged strain. This ensures specificity of measured enzyme activities and would reveal contamination by unspecifically bound enzymes.
Purification and activity assay described here can be performed within two days and the enriched enzymes are stable for at least several months, when stored in aliquots at -80 °C. In conclusion, this protocol provides a relatively simple and cost-effective way to achieve class 1 HDAC complexes for activity measurement and determination of inhibitor efficacy.
The authors have nothing to disclose.
We would like to thank Petra Merschak, Division of Molecular Biology (Biocenter, Medical University of Innsbruck), for her help and support regarding this manuscript. Additionally, we would like to thank the reviewers for their valuable comments.
This work was funded by the Austrian Science Fund (P24803 to SG and P21087 to GB) and by intramural funding (MUI Start, ST201405031 to IB).
10 x SDS-PAGE running buffer | Novex | ||
2-mercaptoethanol (EtSH) | Roth | 4227 | |
25 cm2 cell culture flasks with vent cap | Sarstedt | 833910002 | For spore production |
47 mm vacuum filtration unit | Roth | EYA7.1 | |
AccuFLEX LSC-8000 | HITACHI | – | Scintillation counter |
Acetic acid | Roth | 7332 | |
Anti-Calmodulin Binding Protein Epitope Tag Antibody | Millipore | 07-482 | Used at 1:1333 dilution |
Anti-Rabbit IgG (whole molecule)–Alkaline Phosphatase (AP) antibody | Sigma-Aldrich | A3687 | |
Ball mill | Retsch | 207450001 | Mixer Mill MM 400 |
BCIP/NBT | Promega | S3771 | Color development substrate for AP |
Cell strainer | Greiner | 542040 | |
Cheese cloth for harvesting mycelia | BioRen | H0028 | Topfentuch |
Dimethylsulfoxid (DMSO) | Roth | 4720 | |
EDTA | Prolabo | 20309.296 | |
Ethyl acetate | Scharlau | Ac0155 | |
Freeze Dryer | LABCONCO | 7400030 | FreeZone Triad |
Glycerol | Roth | 3783 | |
HCl | Roth | 4625 | |
IgG resin | GE Healthcare | 17-0969-01 | IgG Sepharose 6 Fast Flow |
Inoculation loops | VWR | 612-2498 | |
KOH | Merck | 5033 | |
Laminar flow cabinet | Thermo Scientific | – | Hera Safe KS |
Mixed Cellulose Esters Membrane Filters | Millipore | GSWP04700 | |
NaCl | Roth | 3957 | |
NaOH | Roth | 6771 | |
Neubauer counting chamber improved | Roth | T728 | |
Novex gel system | Thermo Scientific | For SDS-PAGE | |
Novex Tris-glycine SDS running buffer (10X) | Thermo Scientific | LC2675 | Running buffer for SDS-PAGE |
peqGold protein-marker II | VWR | 27-2010P | Protein ladder used for silver stain |
Peristaltic Pump P-1 | GE Healthcare | 18111091 | |
Pipette controller | Brand | 26302 | accu-jet pro |
Poly-Prep chromatography columns | Bio-Rad | 731-1550 | |
ProSieve QuadColor protein marker | Biozym | 193837 | Prestained protein ladder used for western blot |
Protease inhibitor cocktail tablets | Sigma-Aldrich | 11873580001 | cOmplete, EDTA-free |
Rotary mixer | ELMI | – | Intelli-Mixer RM-2 S |
Rotiszint eco plus | Roth | 0016 | |
RPMI-1640 | Sigma-Aldrich | R6504 | |
Scintillation vials | Greiner | 619080 | |
Sorvall Lynx 4000 | Thermo Scientific | ||
Thermomixer comfort | Eppendorf | ||
TIB32.1 | A. nidulans rpdA::TAP strain. Genotype: alcA(p)::rpdA; veA1; argB2; yA2; pIB32::argB; ArgB+; PyrG+ |
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Trans-Blot Turbo RTA Midi Nitrocellulose Transfer Kit | Bio-Rad | 1704271 | |
Trans-Blot Turbo Transfer System | Bio-Rad | 1704150 | |
Trichostatin A (TSA) | Sigma-Aldrich | T8552 | 5 mM stock in DMSO |
Tris (free base) | Serva | 37190 | |
Tris-HCl | Roth | 9090 | |
Polysorbate 20 | Roth | 9127 | Tween 20 |
Polysorbate 80 | Sigma-Aldrich | P1754 | Tween 80 |
TX-100 | Acros Organics | 215682500 | Triton X-100, Octoxynol-9 detergent |