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Promega Corporation

Cell-Free Expression Systems to Study HDAC Protein Function

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Abstract

Cell-free expression systems offer an alternative pathway to traditional protein production methods. Here we use human histone deacetylase (HDAC) proteins to model how these systems can rapidly make functional proteins. Combined with HaloTag® and Maxwell®16 technologies, cell-free expressed HaloTag® fusion proteins are easily captured on beads from lysate proteins. HaloTag® fluorescent ligands can be used to specifically and covalently attach fluorescent dyes to HaloTag® fusion proteins.

After expression and capture, proteins are assayed for enzymatic activity or used in protein:protein interaction mapping experiments. This integrated project design requires no traditional cloning, little optimization and minimal hands-on time. In this report we describe how we expressed, captured and assayed the enzymatic activity of 11 human HDAC proteins using an integrated proteomics design. In addition to assaying activity, we also used cell-free protein expression to quickly map protein interactions of HDAC2.

Mark Bratz, Brad Hook and Trista Schagat Promega Corporation Publication Date: 2012

Introduction

Expressing and purifying proteins for research can be a long and tedious process. Cell-free expression systems offer an alternative pathway to normal protein production. Using cell-free systems, proteins are expressed in a matter of hours and are ready for either purification or direct functional assays, allowing rapid and easy production of many proteins at one time.

HaloTag® technologies enhance the versatility of cell-free expression by coupling the tag to a protein of interest, allowing detection with fluorescent ligands and capture using affinity resins. Cell-free expressed proteins can be used for functional proteomics, such as enzymatic assays and protein:protein interaction mapping. Coupling HaloTag® technologies with cell-free expression systems decreases the time and complexity of traditional expression and purification methods, allowing novice protein scientists to perform functional assays with little optimization and time.

Histone deacetylases (HDAC) are a class of enzymes that remove acetyl groups from an ε-N-acetyl lysine amino acid on histones (1) . This is important because DNA is wrapped around histones and DNA expression is regulated by acetylation and deacetylation (Figure 1, Panel A). The action of HDAC is opposite to the action of histone acetyltransferase (HAT). Histone tails are normally positively charged due to amine groups on lysine and arginine amino acids. These positive charges help the histone tails to interact with and bind to the negatively charged phosphate groups on the DNA backbone.

10548MAFigure 1. Histone deacetylase (HDAC) activity.

Panel A. Diagram showing gene expression regulated by HDAC and histone acetyltransferase (HAT) proteins. Panel B. Diagram showing HDAC-Glo™ I/II Assay reaction. The enzyme reactions in Panel B, the HDAC-Glo™ I/II Assay, occur virtually simultaneously after the addition of a single reagent.

Panel A. Diagram showing gene expression regulated by HDAC and histone acetyltransferase (HAT) proteins. Panel B. Diagram showing HDAC-Glo™ I/II Assay reaction. The enzyme reactions in Panel B, the HDAC-Glo™ I/II Assay, occur virtually simultaneously after the addition of a single reagent.

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Acetylation, which occurs normally in the cell, neutralizes the positive charges on the histone by changing amines into amides, thus decreasing the ability of the histones to bind to DNA. This decreased binding allows chromatin expansion, permitting genetic transcription to take place. Histone deacetylases remove those acetyl groups, increasing the positive charge of histone tails and encouraging high-affinity binding between the histones and DNA backbone. The increased DNA binding condenses DNA structure, preventing transcription. Histone deacetylase is involved in a series of pathways within the living system and plays an important role in the regulation of gene expression.

The HDAC-Glo™ I/II Assay (Cat.# G6420, G6421) is a single-reagent-addition, homogeneous, luminescent assay that measures the relative activity of histone deacetylase (HDAC) class I and II enzymes from cells, extracts or purified enzyme sources (Figure 1, Panel B). This assay is broadly useful for class I and II enzymes, but sensitivity and performance will vary with catalytic efficiency of particular class I and II isoenzymes. The assay uses an acetylated, live-cell-permeant, luminogenic peptide substrate that can be deacetylated by HDAC activities. Deacetylation of the peptide substrate is measured using a coupled enzymatic system in which a protease in the Developer Reagent cleaves the peptide from aminoluciferin, which is quantified in a reaction using Ultra-Glo™ Recombinant Luciferase. The HDAC-mediated luminescent signal is persistent and proportional to deacetylase activity, allowing batch processing of multiwell plates.

We designed an experimental method to quickly produce and test the function of HDAC proteins (Figure 2). HDAC open reading frame (ORF) DNAs were obtained from the Kazusa Institute as Flexi® vectors. Using the Flexi® System, Transfer, (Cat.# C8820), ORFs were transferred to amino-terminal HaloTag® cell-free Flexi® vectors. Using Magne™ HaloTag® Beads (Cat.# G7281, G7282) in combination with a Maxwell® 16 Instrument, we captured active HDAC protein and assayed using the HDAC-Glo™ I/II Assay. Cell-free expression was also used to produce both bait and prey proteins to test directed protein mapping experiments. These experiments required no traditional cloning, no DNA sequencing, no hands-on purification techniques and no large-scale protein expression and purification. In one day, we were able to express, capture and test the activity of HDAC proteins.

10535MA_700pxFigure 2. Experimental flow diagram.

Red lettering indicates Promega Corporation product, while blue lettering indicates Promega Partner/Affiliate.

Red lettering indicates Promega Corporation product, while blue lettering indicates Promega Partner/Affiliate.

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Methods

Templates
The 11 HDAC open reading frames (ORFs) (Table 1) were obtained from the Kazusa Institute as Flexi® vector constructs. The HDAC ORFs were transferred into a modified pF25K ICE T7 Flexi® Vector (Cat.# L1081) using the Flexi® Vector Systems Technical Manual, #TM254. This modified vector incorporates an amino-terminal HaloTag® into the pF25K ICE T7 Flexi® Vector, which is designed to be used with the TnT® T7 Insect Cell Extract Protein Expression System (Cat.# L1101) (Note: Vector can be obtained through Promega Custom Assay Services, CAS, cas@promega.com). A HaloTag® control protein vector was created by cloning a HaloTag® open reading frame into pF25K ICE T7 Flexi® Vector using the Flexi® Vector System.

Table 1. GenBank® Accession Numbers for HDAC Open Reading Frames (ORFs).
Accession # Available HaloTag® ORF Clone
HDAC1 NM_004964 FHC02563
HDAC2 NM_001527 FHC05328
HCAC3 NM_003883 FHC02559
HDAC4 NM_006037 FHC01664
HDAC5 NM_005474 FHC01982
HDAC6 NM_006044 FHC01133
HDAC7 NM_015401 FHC07742
HDAC8 NM_018486 FHC02809
HDAC9 NM_058176 FHC00124
HDAC10 NM_032019 FHC03143
HDAC11 NM_024827 FHC02775

Protein Expression
All proteins were expressed using the TnT® T7 Insect Cell Extract Protein Expression System (Cat.# L1101). Four micrograms of plasmid was used per 50µl reaction. Incubation was performed at 30°C for 4 hours with shaking at 400rpm using an Eppendorf Thermomixer®.

Manual Bead Capture
Ten microliters of Magne™ HaloTag® Beads (Cat.# G7281,G7282) were prewashed 4 × 1ml with 1X PBS + .005% IGEPAL® in a 1.5ml microcentrifuge tube. After each wash, the microcentrifuge tube containing the beads was placed in a MagneSphere® Technology Magnetic Separation Stand (Cat.# Z5341). The beads were allowed to equilibrate to the magnet then removed from the stand and the wash buffer discarded. Forty-five microliters of cell-free lysate containing expressed HaloTag® fusion proteins was added to the prewashed Magne™ HaloTag® Beads. Binding was allowed to occur for 60 minutes at room temperature while mixing on an Eppendorf Thermomixer®.  A microcentrifuge tube was placed on the magnetic stand and the flowthrough removed. The beads were washed three times with 1ml of 1X PBS + .005% IGEPAL®. After the final wash, 50µl of 1X PBS + .005% IGEPAL® was added to the beads.

Automated Bead Capture
Expressed HaloTag® fusion proteins were captured on beads using the Maxwell® 16 custom method. Empty Maxwell® 16 Cartridges (Custom Cat.# SP1034) were loaded with buffers, Magne™ HaloTag® Beads and lysate expressing the HaloTag® fusion proteins. Cell-free lysate (45µl) containing expressed HaloTag® fusion proteins was added to well 1, and 10µl of prewashed Magne™ HaloTag® Beads was added to well 2. The beads were prewashed four times, 1ml per wash with 1X PBS + .005% IGEPAL®. The cartridges were then inserted into the Maxwell® 16 Instrument (Cat.# AS2000).

To run the custom method, a new firmware version was installed on the instrument with a new method for HaloTag® protein capture (Note: For inquiries into this new firmware, contact Promega Technical Services). For optimal binding and capture, the Maxwell® 16 High Strength LEV Magnetic Rod and Plunger Bar Adaptor (Cat.# SP1070) was used. After capture, the beads were removed from the elution tubes and functional assays were performed.

Functional Assay
The HDAC-Glo™ I/II Assay, (Cat.# G6420; (2) ) was performed as described in the HDAC-Glo I/II Assay and Screening System Technical Manual, #TM335. Fifteen microliters of a 20% slurry of Magne™ HaloTag® Beads with HaloTag®-HDAC protein attached was captured and placed in triplicate in a white 96-well plate. Buffer (50mM Tris [pH 7.5], 150mM NaCl, 2mM KCl, .005% IGEPAL®) was added to a final volume of 100µl. HDAC-Glo™ Reagent (100µl) was added to each well. After brief mixing on a plate shaker and a 30-minute room-temperature incubation, luminescence was measured on a GloMax®-Multi+ Detection System with Instinct™ Software (Cat.# E9032).

Protein:Protein Mapping
HaloTag®-GFP, HaloTag®-HDAC1, HaloTag®-HDAC7, HaloTag®-HDAC 10 and HaloTag®-HDAC2 were expressed using the TnT® T7 Insect Cell Extract Protein Expression System (Cat.# L1101). Reactions were performed as shown in Table 2. Ten microliters of lysates were saved for SDS-PAGE gel analysis. For the bait proteins, 190µl of lysates was bound and captured using the Magne™ HaloTag® Beads. For the prey proteins, 90µl of lysate was labeled with 2µl of HaloTag® TMR Ligand (Cat.# G8251) and incubated at room temperature for 30 minutes. The bait proteins were then divided into 4 reactions and the buffer was removed. Twenty microliters of TMNEN150 buffer (50mM Tris [pH 8.0], 0.5% IGEPAL®, 0.5mM EDTA, 2mM MgCl2, and 150mM NaCl) was added to the particles. The labeled prey reactions (30µl) were added, and the reactions were mixed for 1 hour at 4°C. The beads were washed 3 times with 500µl of TMNEN250 (250mM NaCl) and once with 500µl of TMNEN150. 2X SDS loading dye (25µl) was then added to the settled beads and incubated at 70°C for 10 minutes. Fifteen microliters of each sample was loaded onto a 4–20% SDS-PAGE gel. After separation by electrophoresis at 200V for 1 hour, the gel was imaged using a BioRad XR imager to detect the TMR ligand.

Table 2. Protein:Protein Mapping Reactions.
Template Amount (µg) Lysate (µl) Water (µl)
Bait HDAC2 16 160 to 200
Bait GFP 16 160 to 200
Prey GFP 16 80 to 200
Prey HDAC1 16 80 to 200
Prey HDAC7 16 80 to 200
Prey HDAC10 16 80 to 200

Results

Expression and Capture of HaloTag®-HDAC Proteins
The 11 human HDAC open reading frame DNAs were transferred into a modified pF25K ICE T7 Flexi® Vector (Cat.# L1081) containing an amino-terminal HaloTag® label. This vector enabled production of an amino-terminal HaloTag®-fusion protein when expressed in the TnT® T7 Insect Cell Extract Protein Expression System. The HaloTag®-HDAC proteins expressed in the cell-free expression system were detected by binding the complex to the HaloTag® TMR Ligand, with separation by SDS-PAGE gel (Figure 3, "Total" lanes). All proteins were expressed to detectable levels; however, some protein truncations were observed. Protein levels were normalized based on a HaloTag® standard (Figure 3, “HaloTag® standard curve” lanes). A subset of HDAC open reading frame DNAs were also expressed in the TnT® SP6 High-Yield Wheat Germ Protein Expression System (Cat.# L3260), resulting in yields similar to the TnT® T7 Insect Cell Extract Protein Expression System (data not shown).

10536TA_650pxFigure 3. Expression of HDAC proteins.

Eleven human HDAC proteins were expressed in a cell-free system as HaloTag® fusion proteins and captured on Magne™ HaloTag® Beads (Cat.# G7281). HaloTag® fusions in the expression lysates "Total") and in the flowthrough ("Unbound") were labeled using the HaloTag® TMR Ligand and separated by SDS-PAGE gel. Proteins were detected using fluorescence. HaloTag® Standard Protein (Cat.# G4491) was labeled and used to normalize protein expression. Some protein truncation is seen on the gel (e.g., HaloTag®-HDAC2).

Eleven human HDAC proteins were expressed in a cell-free system as HaloTag® fusion proteins and captured on Magne™ HaloTag® Beads (Cat.# G7281). HaloTag® fusions in the expression lysates "Total") and in the flowthrough ("Unbound") were labeled using the HaloTag® TMR Ligand and separated by SDS-PAGE gel. Proteins were detected using fluorescence. HaloTag® Standard Protein (Cat.# G4491) was labeled and used to normalize protein expression. Some protein truncation is seen on the gel (e.g., HaloTag®-HDAC2).

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We used the Maxwell® 16 Instrument as a method to purify the HaloTag®-fusion proteins with little hands-on work. Adapting the instrument to fit our needs for protein capture on beads involved a few steps (see the methods section for more details). A custom method was created that ends with the magnetic beads in the elution tubes. Cartridge fill conditions were optimized with the maximum wash buffer volumes added to each wash well. Cell-free expression reactions were loaded directly into the first well of the cartridge. After the method was complete, flowthrough from the first well was taken to ascertain how efficiently the proteins were binding to the beads (Figure 3, "Unbound' lanes). We observed very little HaloTag® fusion protein in well 1 (flowthough), indicating complete binding of the expressed HaloTag®-HDAC fusion proteins to the magnetic beads. We also observed no significant bead loss in wells 1–8, as all the beads were now in the elution tube (data not shown).

Comparison of Manual and Maxwell® 16 Methods for Capture of HaloTag® Fusion Proteins
To ensure that the HaloTag® fusion proteins captured by the Maxwell® 16 method were as highly purified from contaminating proteins in the cell-free lysate as in a manual method, we performed a direct comparison of the bead-bound HaloTag®-HDAC11 protein. HaloTag®-HDAC11 was expressed in a cell-free system and captured on beads by both the Maxwell® 16 method and by a manual method.

HaloTag® control and lysate-only reactions were also performed to assay for background activity and carryover from lysate deacetylases. For the best possible comparison, both methods used the same initial cell-free reaction, buffers, amount of Magne™ HaloTag® Beads, binding and wash times, buffer volumes, and elution volume. For the manual method, beads were separated from buffer using a 1.5ml tube magnetic stand. Total lysate and bead flowthrough ("Unbound") from HaloTag®-HDAC11, HaloTag® control and lysate control were labeled with the HaloTag® TMR Ligand and separated using an SDS-PAGE gel (Figure 4, Panel A). Both HaloTag®-HDAC11 and HaloTag® control protein were expressed at high levels in the cell-free systems. Both methods showed significant depletion of the fusion protein in the flowthrough; however, some HaloTag®-HDAC11 was observed in the manual method.

After capture, the bead-bound HaloTag®-HDAC11 proteins along with the control beads were assayed using the HDAC-Glo™ I/II Assay (Figure 4, Panel B). Both HaloTag® control and lysate only samples displayed background activity from nonspecific binding of lysate deacetylases. Both capture methods performed similarly with the Maxwell® 16 method, resulting in a 24.6 ± 0.8-fold increase over control and the manual method resulting in a 22.1 ± 0.1-fold increase over control. This indicates that the Maxwell® 16 method is a viable alternative to manual purification of proteins. To further scale-up HaloTag® fusion protein capture, one can use a high-throughput automated system as demonstrated by Ohana, Vidugiris and Encell  (3) .

10537TA_600pxFigure 4. Capture of HaloTag®-HDAC11 on beads using both manual and Maxwell® 16 methods.

Panel A. SDS-PAGE gel showing TMR-labeled HaloTag®-HDAC11 captured on beads using both manual and Maxwell® 16 methods. Flowthrough is the unbound protein from the capture method. Panel B. Graph displaying HDAC activity using the HDAC-Glo™ I/II  Assay. Bars and error results from replicates of three.

Panel A. SDS-PAGE gel showing TMR-labeled HaloTag®-HDAC11 captured on beads using both manual and Maxwell® 16 methods. Flowthrough is the unbound protein from the capture method. Panel B. Graph displaying HDAC activity using the HDAC-Glo™ I/II  Assay. Bars and error results from replicates of three.

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Optimization of Capture Buffer
To ensure that we captured the HDAC proteins in a buffer that allows for maximal activity, while reducing background deacetylase activity found in all cell-free lysates, we performed a buffer optimization experiment in which 8 different buffers were tested in the capture of one HaloTag®-HDAC fusion protein. HaloTag®-HDAC1 and HaloTag® control protein were expressed in a cell-free system and split into eight Maxwell® 16 Cartridges each. Table 3 lists the different buffers that were used in the protein capture methods. Buffer 1 was used to wash the Magne™ HaloTag® Beads before addition to the Maxwell® 16 cartridges and as a gentle capture buffer. The remaining buffers increase both salt and detergent content, resulting in increasing stringency. Buffer 9 was used as the universal final wash and elution buffer due to its lowered salt concentration.

Table 3. Composition of Buffers.
Buffer NaCl KCl Detergent
Buffer 1 1X PBS -- 25mM 0.005% IGEPAL®
Buffer 2 50mM Tris (pH 7.5) 500mM 25mM 0.005% Triton®
Buffer 3 50mM Tris (pH 7.5) 250mM 25mM 0.005% Triton®
Buffer 4 50mM Tris (pH 7.5) 500mM 25mM 1% Triton®
Buffer 5 50mM Tris (pH 7.5) 250mM 25mM 1% Triton®
Buffer 6 50mM Tris (pH 7.5) 500mM 25mM 1% IGEPAL®
Buffer 7 50mM Tris (pH 7.5) 250mM 25mM 1% IGEPAL®
Buffer 8 1X PBS -- -- 1% Triton®
Buffer 9 50mM Tris (pH 7.5) 150mM 2mM 0.005% IGEPAL®

After capture, the captured HaloTag®-HDAC and HaloTag® control beads were assayed for activity using the HDAC-Glo™ I/II Assay (Figure 5). When comparing RLU values, HDAC1 activities varied from approximately 2.8 × 106 to approximately 1.4 × 106 in the different buffers; however, the HaloTag® control activities also widely varied (Figure 5, Panel A). When comparing HDAC1 activities as a fold increase over the HaloTag® control activities, Buffer 5 resulted in the greatest fold increase over control, approximately 30-fold as compared to Buffer 3, which had only an approximately 11-fold increase (Figure 5, Panel B). Buffer 5 did not produce protein with the highest overall relative light units (RLU); however, it significantly reduced the background signal in the HaloTag® control, which in turn produced the greatest fold increase. All further experiments used Buffer 5 as the main wash buffer.

10538MA_550pxFigure 5. Buffer optimization using Maxwell® 16 protein capture method.

Graphs depict HDAC activity using HDAC-Glo™ I/II Assay from Maxwell® 16 buffer optimization experiment. Numbers below the graph refer to buffer composition found in Table 3. Panel A. Graph showing relative light units (RLU). Panel B. Graph showing fold increase over HaloTag® control protein. Bars and error result from replicates of three.

Graphs depict HDAC activity using HDAC-Glo™ I/II Assay from Maxwell® 16 buffer optimization experiment. Numbers below the graph refer to buffer composition found in Table 3. Panel A. Graph showing relative light units (RLU). Panel B. Graph showing fold increase over HaloTag® control protein. Bars and error result from replicates of three.

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HDAC Protein Activity
The 11 HaloTag®-HDAC proteins captured on beads using the Maxwell® 16 method, were tested for activity using the HDAC-Glo™ I/II Assay (Figure 6, Panel A). HDAC1, 2 and 3 had the greatest activity, whereas HDAC4 had the lowest activity. While HDAC4 had the lowest activity, it also was shown to be truncated in Figure 2, which could account for the drop in activity. Most HDAC proteins had about a 20-fold increase in activity over the HaloTag® control. HaloTag®-HDAC proteins expressed in the SP6 Wheat Germ System and captured on beads exhibited similar activity using the HDAC-Glo™ I/II Assay to the proteins expressed in the TnT® T7 Insect Cell Extract Protein Expression System (data not shown).

10539MA_550pxFigure 6. Activity of 11 human HDAC proteins expressed in cell-free expression system.

The graph displays HDAC activity using the HDAC-Glo™ I/II Assay on 11 human HDAC proteins expressed using a cell-free system. The graph plots fold increase over HaloTag® control protein. Bars and error result from replicates of three.

The graph displays HDAC activity using the HDAC-Glo™ I/II Assay on 11 human HDAC proteins expressed using a cell-free system. The graph plots fold increase over HaloTag® control protein. Bars and error result from replicates of three.

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Protein:Protein Interactions
Analyzing protein:protein interactions is an important part of functional proteomics. Most proteins function in complexes; therefore, identifying protein:protein interactions is critical to dissecting protein function. HDAC2 has been shown to physically bind to HDAC1, forming a nuclear co-repressor complex (4) . Using immunoprecipitation experiments, HDAC2 has also been shown to be associated in a complex with HDAC10; however, direct interaction was not tested  (5) . Using direct protein:protein pull-down experiments, HDAC7 has been shown to interact with some HDAC proteins but not HDAC2 (6) .

To test protein:protein interactions of HDAC proteins expressed in cell-free, we performed directed binding experiments with HaloTag®-HDAC2 to HDAC1, HDAC10 and HDAC7 (Figure 7, Panel A). HaloTag®-HDAC2 and control protein HaloTag®-GFP were expressed in a cell-free system, and captured on beads using our Maxwell® 16 method. These bait proteins will remain bead-bound in the experiment. HaloTag®-HDAC1, HaloTag®-HDAC7, HaloTag®-HDAC10 and HaloTag®-GFP were expressed cell-free and labeled with excess HaloTag® TMR Ligand. These prey proteins were incubated with the bait proteins, then complexes were washed and eluted. Samples were separated using SDS-PAGE gel and detected using fluorescence (Figure 7, Panel B). All prey proteins were expressed and labeled (Figure 7, Panel B, “Prey Input” lanes). Positive protein:protein interactions were observed between HDAC2/HDAC1 and HDAC2/HDAC10 but not HDAC2/HDAC7. As previously reported, HDAC2 interacted with HDAC1 but not HDAC7 (6) . We were able to confirm that HDAC2 not only associates with HDAC10 as previously reported, but forms a direct protein:protein interaction. Using only cell-free expressed proteins, we were able to identify protein:protein interactions in a one-day experiment.

10540TA_650pxFigure 7. Using cell-free expression to map HDAC2 interactions.

Panel A. Protein:protein interaction scheme. Orange circles represent TMR-labeled, HaloTag®-fused HDAC1, HDAC7, HDAC10 or GFP. Pink stars represent TMR fluorescence. Black circles represent Magne™ HaloTag® Beads. HT = HaloTag®. Panel B. SDS-PAGE of protein:protein interactions. The labels above the gels indicate the Bait proteins, and labels below the gel indicate Prey proteins. Red lettering represents no interaction, and green lettering represents positive interaction. The diagram below the gel represents the interaction map between the proteins tested.

Panel A. Protein:protein interaction scheme. Orange circles represent TMR-labeled, HaloTag®-fused HDAC1, HDAC7, HDAC10 or GFP. Pink stars represent TMR fluorescence. Black circles represent Magne™ HaloTag® Beads. HT = HaloTag®. Panel B. SDS-PAGE of protein:protein interactions. The labels above the gels indicate the Bait proteins, and labels below the gel indicate Prey proteins. Red lettering represents no interaction, and green lettering represents positive interaction. The diagram below the gel represents the interaction map between the proteins tested.

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Conclusions

Using cell-free expression systems in combination with HaloTag® technologies, we expressed and captured active HDAC proteins on beads. The Maxwell® 16 Instrument proved to be a power tool for rapid hands-free capture of HaloTag®-HDAC proteins expressed in cell-free. The HDAC-Glo™ I/II Assay provided a sensitive and robust assay for quantitation of HDAC activity. Cell-free expression was also used to produce both bait and prey proteins to test directed protein mapping experiments, which resulted in positive interactions between HDAC1 and HDAC2 and between HDAC2 and 10. These experiments required no traditional cloning, no DNA sequencing, no hands-on capture techniques and no large-scale protein expression and purification. The speed of cell-free expression and HaloTag® technology assay products allowed expression, purification and functional testing of HDAC proteins in a one-day experiment.

Editors Note: Promega does not currently sell Maxwell® 16 cartridges containing Magne™ HaloTag® Beads. However, the Magne™ HaloTag® Beads can be used for capture of HaloTag®-HDAC proteins manually.

References

  1. Peserico, A. and Simone, C. (2011) Physical and functional HAT/HDAC interplay regulates protein acetylation balance. J. Biomed. Biotechnol. 2011, 1–10.
  2. Halley, F. et al. (2011) A Bioluminogenic HDAC Activity Assay: Validation and Screening. J. Biomol. Scr. epub ahead of print.
  3. Ohana, R.F., et al. (2012) Efficient high-throughput protein purification using the Magne™ HaloTag® Beads. PubHub Accessed March 28 2012.
  4. Hassig, C.A. et al. (1998) A role for histone deacetylase activity in HDAC1-mediated transcriptional repression. Proc. Natl. Acad. Sci. USA 95, 3519–24.
  5. Fischer, D.D. et al. (2002) Isolation and characterization of a novel class II histone deacetylase, HDAC10. J. Biol. Chem. 277, 6656–66.
  6. Fischle, W. et al. (2001) Human HDAC7 histone deacetylase activity is associated with HDAC3 in vivo. J. Biol. Chem. 276, 35826–35.

How to Cite This Article

Bratz, M., Hook, B. and Schagat, T. Cell-Free Expression Systems to Study HDAC Protein Function. [Internet] 2012. [cited: year, month, date]. Available from: http://www.promega.com/resources/pubhub/cell-free-expression-systems-to-study-hdac-protein-function-article/

Bratz, M., Hook, B. and Schagat, T. Cell-Free Expression Systems to Study HDAC Protein Function. Promega Corporation Web site. http://www.promega.com/resources/pubhub/cell-free-expression-systems-to-study-hdac-protein-function-article/ Updated 2012. Accessed Month Day, Year.

Flexi, GloMax, HaloTag, MagneSphere, Maxwell and TnT are registered trademarks of Promega Corporation.

HDAC-Glo, Instinct, Magne, PureYield and Ultra-Glo are trademarks of Promega Corporation.

GenBank is a registered trademark of U.S. Department of Health and Human Services. IGEPAL is a registered trademark of Rhone-Poulenc AG Co. Thermomixer is a registered trademark of Eppendorf AG. Triton is a registered trademark of Union Carbide Chemicals & Plastics Technology Corporation.



Figures

10548MAFigure 1. Histone deacetylase (HDAC) activity.

Panel A. Diagram showing gene expression regulated by HDAC and histone acetyltransferase (HAT) proteins. Panel B. Diagram showing HDAC-Glo™ I/II Assay reaction. The enzyme reactions in Panel B, the HDAC-Glo™ I/II Assay, occur virtually simultaneously after the addition of a single reagent.

Panel A. Diagram showing gene expression regulated by HDAC and histone acetyltransferase (HAT) proteins. Panel B. Diagram showing HDAC-Glo™ I/II Assay reaction. The enzyme reactions in Panel B, the HDAC-Glo™ I/II Assay, occur virtually simultaneously after the addition of a single reagent.

/~/media/images/resources/figures/10500-10599/10548ma.jpg?la=en
10535MA_700pxFigure 2. Experimental flow diagram.

Red lettering indicates Promega Corporation product, while blue lettering indicates Promega Partner/Affiliate.

Red lettering indicates Promega Corporation product, while blue lettering indicates Promega Partner/Affiliate.

/~/media/images/resources/figures/10500-10599/10535ma_700px.jpg?la=en
10536TA_650pxFigure 3. Expression of HDAC proteins.

Eleven human HDAC proteins were expressed in a cell-free system as HaloTag® fusion proteins and captured on Magne™ HaloTag® Beads (Cat.# G7281). HaloTag® fusions in the expression lysates "Total") and in the flowthrough ("Unbound") were labeled using the HaloTag® TMR Ligand and separated by SDS-PAGE gel. Proteins were detected using fluorescence. HaloTag® Standard Protein (Cat.# G4491) was labeled and used to normalize protein expression. Some protein truncation is seen on the gel (e.g., HaloTag®-HDAC2).

Eleven human HDAC proteins were expressed in a cell-free system as HaloTag® fusion proteins and captured on Magne™ HaloTag® Beads (Cat.# G7281). HaloTag® fusions in the expression lysates "Total") and in the flowthrough ("Unbound") were labeled using the HaloTag® TMR Ligand and separated by SDS-PAGE gel. Proteins were detected using fluorescence. HaloTag® Standard Protein (Cat.# G4491) was labeled and used to normalize protein expression. Some protein truncation is seen on the gel (e.g., HaloTag®-HDAC2).

/~/media/images/resources/figures/10500-10599/10536ta_650px.jpg?la=en
10537TA_600pxFigure 4. Capture of HaloTag®-HDAC11 on beads using both manual and Maxwell® 16 methods.

Panel A. SDS-PAGE gel showing TMR-labeled HaloTag®-HDAC11 captured on beads using both manual and Maxwell® 16 methods. Flowthrough is the unbound protein from the capture method. Panel B. Graph displaying HDAC activity using the HDAC-Glo™ I/II  Assay. Bars and error results from replicates of three.

Panel A. SDS-PAGE gel showing TMR-labeled HaloTag®-HDAC11 captured on beads using both manual and Maxwell® 16 methods. Flowthrough is the unbound protein from the capture method. Panel B. Graph displaying HDAC activity using the HDAC-Glo™ I/II  Assay. Bars and error results from replicates of three.

/~/media/images/resources/figures/10500-10599/10537ta_600px.jpg?la=en
10538MA_550pxFigure 5. Buffer optimization using Maxwell® 16 protein capture method.

Graphs depict HDAC activity using HDAC-Glo™ I/II Assay from Maxwell® 16 buffer optimization experiment. Numbers below the graph refer to buffer composition found in Table 3. Panel A. Graph showing relative light units (RLU). Panel B. Graph showing fold increase over HaloTag® control protein. Bars and error result from replicates of three.

Graphs depict HDAC activity using HDAC-Glo™ I/II Assay from Maxwell® 16 buffer optimization experiment. Numbers below the graph refer to buffer composition found in Table 3. Panel A. Graph showing relative light units (RLU). Panel B. Graph showing fold increase over HaloTag® control protein. Bars and error result from replicates of three.

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10539MA_550pxFigure 6. Activity of 11 human HDAC proteins expressed in cell-free expression system.

The graph displays HDAC activity using the HDAC-Glo™ I/II Assay on 11 human HDAC proteins expressed using a cell-free system. The graph plots fold increase over HaloTag® control protein. Bars and error result from replicates of three.

The graph displays HDAC activity using the HDAC-Glo™ I/II Assay on 11 human HDAC proteins expressed using a cell-free system. The graph plots fold increase over HaloTag® control protein. Bars and error result from replicates of three.

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10540TA_650pxFigure 7. Using cell-free expression to map HDAC2 interactions.

Panel A. Protein:protein interaction scheme. Orange circles represent TMR-labeled, HaloTag®-fused HDAC1, HDAC7, HDAC10 or GFP. Pink stars represent TMR fluorescence. Black circles represent Magne™ HaloTag® Beads. HT = HaloTag®. Panel B. SDS-PAGE of protein:protein interactions. The labels above the gels indicate the Bait proteins, and labels below the gel indicate Prey proteins. Red lettering represents no interaction, and green lettering represents positive interaction. The diagram below the gel represents the interaction map between the proteins tested.

Panel A. Protein:protein interaction scheme. Orange circles represent TMR-labeled, HaloTag®-fused HDAC1, HDAC7, HDAC10 or GFP. Pink stars represent TMR fluorescence. Black circles represent Magne™ HaloTag® Beads. HT = HaloTag®. Panel B. SDS-PAGE of protein:protein interactions. The labels above the gels indicate the Bait proteins, and labels below the gel indicate Prey proteins. Red lettering represents no interaction, and green lettering represents positive interaction. The diagram below the gel represents the interaction map between the proteins tested.

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