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Immunoaffinity purification of the functional 20S proteasome from human cells via transient overexpression of specific proteasome subunits
Protein Expression and Purification 97 (2014) 37–43
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Immunoaffinity purification of the functional 20S proteasome from human cells via transient overexpression of specific proteasome subunits Veronika A. Livinskaya a,b, Nickolai A. Barlev a,c, Andrey A. Nikiforov a,b,⇑ a b c
Institute of Cytology, Russian Academy of Science, Tikhoretsky ave. 4, 194064 Saint Petersburg, Russia Institute of Nanobiotechnologies, Saint Petersburg State Polytechnical University, Polytechnicheskaya 29, 195251 Saint Petersburg, Russia Department of Biochemistry, University of Leicester, Leicester LE1 9HN, United Kingdom
a r t i c l e
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Article history: Received 15 December 2013 and in revised form 15 February 2014 Available online 28 February 2014 Keywords: Immunoaffinity purification 20S proteasome 3xFLAG peptide PSMB5 PSMA5 PSMA3
a b s t r a c t The proteasome is a multi-subunit proteolytic complex that plays a central role in protein degradation in all eukaryotic cells. It regulates many vital cellular processes therefore its dysfunction can lead to various pathologies including cancer and neurodegeneration. Isolation of enzymatically active proteasomes is a key step to the successful study of the proteasome regulation and functions. Here we describe a simple and efficient protocol for immunoaffinity purification of the functional 20S proteasomes from human HEK 293T cells after transient overexpression of specific proteasome subunits tagged with 3xFLAG. To construct 3xFLAG-fusion proteins, DNA sequences encoding the 20S proteasome subunits PSMB5, PSMA5, and PSMA3 were cloned into mammalian expression vector pIRES-hrGFP-1a. The corresponding recombinant proteins PSMB5-3xFLAG, PSMA5-3xFLAG, or PSMA3-3xFLAG were transiently overexpressed in human HEK 293T cells and were shown to be partially incorporated into the intact proteasome complexes. 20S proteasomes were immunoprecipitated from HEK 293T cell extracts under mild conditions using antibodies against FLAG peptide. Isolation of highly purified 20S proteasomes were confirmed by SDS–PAGE and Western blotting using antibodies against different proteasome subunits. Affinity purified 20S proteasomes were shown to possess chymotrypsin- and trypsin-like peptidase activities confirming their functionality. This simple single-step affinity method of the 20S proteasome purification can be instrumental to subsequent functional studies of proteasomes in human cells. Ó 2014 Elsevier Inc. All rights reserved.
Introduction The ubiquitin–proteasome system (UPS)1 catalyzes the selective degradation of short-lived regulatory proteins and proteins with abnormal conformation in eukaryotic cells. UPS regulates many vital cellular processes such as cell cycle progression, apoptosis, DNA repair, immune responses, protein quality control therefore its ⇑ Corresponding author at: Institute of Cytology, Russian Academy of Science, Tikhoretsky ave. 4, 194064, Saint Petersburg, Russia. Tel.: +7 911 9050330; fax: +7 812 2973541. E-mail address: [email protected] (A.A. Nikiforov). 1 Abbreviations used: UPS, ubiquitin-proteasome system; RP, regulatory particle; HIF-1, hypoxia-inducible factor-1; SRC-3, steroid receptor coactivator-3; HEK, human embryonic kidney; Suc-LLVY-AMC, Succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosine-4methylcoumaryl-7-amide; Boc-LRR-AMC, t-Butyloxycarbonyl-L-leucyl-L-arginyl-Larginine-4-methylcoumaryl-7-amide; AMC, 7-amino-4-methylcoumarin; LB, Luria– Bertani medium; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; HBS, hepes buffered saline; RT, room temperature; PBS, phosphate buffered saline; PMSF, phenylmethylsulfonyl fluoride; WB, washing buffer. http://dx.doi.org/10.1016/j.pep.2014.02.011 1046-5928/Ó 2014 Elsevier Inc. All rights reserved.
dysfunction can lead to various pathologies like cancer or neurodegeneration [1,2]. The 26S proteasome is an ATP-dependent multisubunit protease that degrades polyubiquitinated proteins to small peptides. It is composed of a catalytic 20S core complex, also called the 20S proteasome, and one or two 19S regulatory particles (RP). The 20S proteasome is a 700 kDa barrel-shaped protein complex consisting of four stacked heptameric rings. Each of the two inner rings is composed of seven different b-subunits (PSMB1–7), three of which (PSMB5, PSMB6 and PSMB7) represent chymotrypsin-like, trypsin-like, and caspase-like proteolytic activities of the 20S proteasome. Seven homologous but not identical a-subunits (PSMA1–7) compose each of the two outer rings which regulate substrate access to proteolytic chamber of the 20S proteasome and binding of regulatory complexes. The 19S RP is a 700 kDa protein complex consisting of about 20 various subunits. 19S RP selects substrates for degradation by binding polyubiquitin chains of target proteins which are then deubiquitinated, unfolded and translocated to the proteolytic chamber of the 20S proteasome for breakdown [2–4]. Importantly,
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proteasomes undergo ubiquitinylation by themselves, which affects their proteolytic activities [5]. Besides ubiquitin-dependent protein degradation, 26S proteasome, as well as 20S proteasome alone, also controls ubiquitinindependent cleavage of various regulatory proteins. Tumor suppressor p53, inhibitor of cyclin-dependent kinases p21WAF1/CIP1, transcription factors c-Fos, Fra-1 and HIF-1, calmodulin, steroid receptor coactivator-3 (SRC-3) and many other proteins have been shown to be regulated by ubiquitin-independent proteasomal degradation, mostly via interaction with the PSMA3 subunit of the 20S complex [6–9]. In addition, 20S proteasome catalyzes endoproteolytic cleavage of polypeptide precursors resulting in their maturation [6]. Besides the proteolytic activities 20S proteasomes were shown to possess non-canonical endoribonuclease activity [10,11]. Proteasome purification is a crucial step in the majority of studies focused on proteasome regulation and functions. Conventional proteasome purification usually takes several days and includes three common steps: ultracentrifugation (or gel filtration chromatography), ion exchange chromatography, and hydrophobic interaction chromatography [12–15]. This procedure is performed under strict experimental conditions, such as exposure to high salt concentrations, which can lead to loss of proteasome-associated proteins during purification. Since the last decade, faster and easier affinity-based strategy for proteasome purification has been developed. This approach is based on single-step affinity chromatography after overexpression of epitope-tagged proteasome subunits in yeast or human cells [14,16–18]. Affinity purification of 20S or 26S proteasome is conducted under mild conditions which preserve weak protein–protein interactions. Therefore this method is a powerful tool for purification with further characterization of proteasome-associated proteins [17–20].To date, a substantial body of evidence has been accumulated suggesting that the 20S proteasome exerts important biological functions on its own, independently of the 19S complex [21,22]. Therefore, an implementation of novel robust and simple biochemical approaches to purify functional 20S proteasomes is well justified. Here we present the development of a simple and efficient immunoaffinity method for purification of an enzymatically active 20S proteasomes from cultured human HEK 293T cells via transient overexpression of 3xFLAG-tagged proteasome subunits PSMB5, PSMA5, or PSMA3. Highly purified tagged 20S proteasomes were obtained by immunoprecipitation from cell extracts using monoclonal antibodies to FLAG peptide.
Materials and methods Materials Escherichia coli DH10 cells were used for cloning and plasmid amplification. Human embryonic kidney (HEK) 293T cell line was obtained from the cell culture bank of the Institute of Cytology, RAS (St. Petersburg, Russia). pIRES-hrGFP-1a plasmid was purchased from Agilent Technologies. ANTI-FLAG M2 Magnetic Beads were purchased from Sigma–Aldrich. The following antibodies were used for Western blotting: mouse anti-FLAG (M2), HRP-conjugated goat anti-rabbit and rabbit anti-mouse antibodies (Sigma–Aldrich); rabbit anti-PSMA3, rabbit anti-PSMA5, and rabbit anti-PSMB5 were custom generated in the laboratory [23]; mouse anti-PSMA1, mouse anti-PSMA4, mouse anti-PSMB6, mouse antiPSMB7, and mouse anti-PSMD6 (Enzo Life Sciences). DNA modifying and restriction enzymes were obtained from Fermentas or New England BioLabs. Oligonucleotides were synthesized by Sigma–Aldrich. To measure the peptidase activity of proteasomes the following fluorogenic peptides were used: t-Butyloxycarbonyl-L-leucyl-L-arginyl-L-arginine-4-methylcoumaryl-7-amide
(Boc-LRR-AMC) and Succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosine-4methylcoumaryl-7-amide (Suc-LLVY-AMC) (all purchased from Enzo Life Sciences). Cloning and generation of mammalian expression vectors To generate expression vectors with 3xFLAG-tagged PSMA3, PSMA5 and PSMB5 proteasome subunits in human cells the corresponding DNA sequences (GenBank Accession Nos. NM_002788, NM_002790, and NM_002797, respectively) were amplified from a cDNA library of human K562 cells using the following primers: PSMA3-F 5’-CGTAGGATCCATGAGCTCAATC-3’, PSMA3-R 5’-GTAA CTCGAGCATATTATCATCATC-3’ containing the BamH1 and Xho1 restriction sites, respectively; PSMA5-F 5’-GCCGAATTC AATGTTTCT TACCCGGTC-3’, PSMA5-R 5’-GCGGTCGACGAATGTC CTTGATAACC3’ containing the EcoR1 and SalI restriction sites, respectively; PSMB5-F: 5’-AGTCGAATTCAAT GGCGCTTGCCAGC-3’, PSMB5-R: 5’-GATACTCGAGGGGGGTAGAGCCAC-3’ containing the EcoR1 and Xho1 restriction sites, respectively. Amplified fragments of PSMA3, PSMA5 and PSMB5 were then inserted into the plasmid pIRES-hrGFP-1a digested with the matching pairs of restriction enzymes. Competent E. coli DH10 cells were transformed with obtained recombinant plasmids pIRES-hrGFP-1a-PSMA3, pIRES-hrGFP-1aPSMA5, or pIRES-hrGFP-1a-PSMB5 and plated onto LB agar plates supplemented with ampicillin (100 lg/ml). Transformants were screened by PCR and were subsequently verified by the restriction analysis. The accuracy of cloning was confirmed by sequencing. Cell culture and transfection HEK 293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS (Gibco), 2 mM L-glutamine and 20 lg/ml gentamicin. The cells were cultured at 37 °C in a humidified atmosphere of 5% CO2. Transient transfection of HEK 293T cells with vectors pIRES-hrGFP-1a-PSMA3, pIRES-hrGFP-1a-PSMA5, or pIRES-hrGFP1a-PSMB5 was done using the calcium phosphate method. Cells were seeded in a 100 mm culture plate and then grown to 50–70% confluence. One hour before transfection fresh medium (10 ml) was added to cells. Pure plasmid DNA (8–10 lg) was dissolved in 600 ll of 250 mM CaCl2 solution. Then the mixture was carefully added to 600 ll of 2 HBS buffer (50 mM Hepes, 10 mM KCl, 1.5 mM Na2HPO4, and 280 mM NaCl, pH 7.05) with concomitant vortexing. After 20 min incubation at room temperature (RT) the mixture (final volume of 1200 ll) was added dropwise onto the culture plate. The culture plate was carefully shaken to equally disperse the calcium phosphate precipitates. Cells were incubated for 24–96 h at 37 °C, and then the whole cell extract was prepared and used for immunoprecipitation or Western blotting analysis. Immunoaffinity purification of 20S proteasome HEK 293T cells, grown in a four 100 mm culture plates (5x10E6 cells per plate), were transfected with vectors pIRES-hrGFP-1aPSMB5, pIRES-hrGFP-1a-PSMA5, or pIRES-hrGFP-1a-PSMA3 encoding fusion proteins PSMB5-3xFLAG, PSMA5-3xFLAG, or PSMA33xFLAG, respectively. 24 h after transfection cells from four 100 mm plates (for each transfection) were plated onto six 150 mm plates and incubated for 72 h at 37 °C. 96 h after transfection cells were washed twice with ice-cold PBS and lysed in the lysis buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 0.3% Triton X-100, and 0.5 mM PMSF) by agitation for 40 min at 4 °C. The resulting cell lysate was clarified by centrifugation at 20,000g for 30 min at 4 °C, and the supernatant was collected. The cell extract
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was incubated with 100 ll of Anti-FLAG M2 magnetic beads overnight at 4 °C under gentle rocking. The beads were then washed five times with 5 ml of washing buffer (WB) containing 50 mM Tris (pH 7.5), 150 mM NaCl, and 0.5 mM PMSF. Immunoprecipitated proteins were eluted at 4 °C with 500 ll of WB supplemented with 3[FLAG peptide (300 lg/ml). 3[FLAG peptide was then removed by serial concentration on 10 kDa concentrators (Millipore). The immunoaffinity-purified protein complexes were characterized by SDS–PAGE, Western blotting and proteasome activity, as described below. Protein determination, SDS–PAGE, and western blotting Protein concentration of samples was determined using BCA protein assay kit (Pierce) according to the manufacturer’s protocol. All samples were prepared by boiling in a Laemmli buffer (62.5 mM Tris–HCl (pH 6.8), 2% SDS, 0.05% bromophenol blue, 10% glycerol, and 100 mM DTT) for 7 min. Then proteins were resolved by 12% SDS–polyacrylamide gel electrophoresis (SDS–PAGE) followed by visualization using Coomassie blue staining. For Western blotting, proteins were transferred onto a PVDF membrane after separation by SDS–PAGE. The obtained membrane was blocked with PBS supplemented with 5% (w/v) non-fat dry milk and 0.1% (v/v) Tween 20 for 1 h at RT or overnight at 4 °C. Following blocking, the membrane was incubated with appropriate primary and HRP-conjugated secondary antibodies was performed in PBS supplemented with 1% (w/v) non-fat dry milk and 0.1% (v/v) Tween 20 for 1 h at RT. The membranes were washed six times in PBS supplemented with 0.1% (v/v) Tween 20 after incubations with primary and secondary antibodies. ECL detection was performed with SuperSignal system (Thermo Fisher Scientific) according to the manufacturer’s protocol. Proteasome activity assay The chymotrypsin- and trypsin-like peptidase activities of purified 20S proteasomes were calculated based on the efficiency of hydrolysis of the respective fluorogenic substrates Suc-LLVY-AMC and Boc-LRR-AMC. The assay was conducted in the buffer containing 150 lM Suc-LLVY-AMC or 480 lM Boc-LRR-AMC, 50 mM Tris–HCl (pH 7.5), 5 mM MgCl2, 40 mM KCl, and 1 mM DTT at 37 °C for 1 h. The liberated 7-amino-4-methylcoumarin (AMC) was measured fluorimetrically (excitation at 365 nm, emission at 440 nm). For the control of specificity 20S proteasomes were treated with a proteasome inhibitor MG132 or mock-treated with DMSO for 20 min at RT before adding the substrates. The specific activity of the purified 20S proteasome was calculated using AMC-standard curve and presented as the pmol of released AMC per 1 h per 1 lg of proteasome. Glycerol density gradient centrifugation HEK 293T cells were transfected with vectors pIRES-hrGFP-1aPSMB5, pIRES-hrGFP-1a-PSMA5, or pIRES-hrGFP-1a-PSMA3. 96 h after transfection cells were washed twice with ice-cold PBS and lysed in the lysis buffer (50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 0.1% Triton X-100, and 0.5 mM PMSF) by agitation for 40 min at 4 °C. The resulting cell lysates were clarified by centrifugation at 25,000g for 30 min at 4 °C, and the supernatants were collected. Soluble extracts from control and transfected cells were loaded onto a 5 to 30% glycerol density gradient, followed by centrifugation at 80,000 g for 17 h at 4 °C. Eleven fractions were collected from the top of the gradients, and the proteins were precipitated with 80% ethanol. Then precipitated proteins were dissolved in
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Laemmli buffer, subjected to SDS–PAGE and analyzed by Western blotting.
Results and discussion The fusion of a protein of interest with affinity tag is a commonly used method for purification of recombinant proteins. Immunoaffinity purification of fusion proteins using antibodies against tag peptide is performed under mild non-denaturing conditions that preserve physiological protein–protein associations and thus can be used for isolation of native protein complexes from mammalian cells [24]. This study aimed to establish a simple and efficient method for immunoaffinity purification of functional 20S proteasomes from human HEK 293T cells after transient overexpression of 3xFLAG-tagged proteasome subunits using antibodies to FLAG peptide. Although TEV protease cleavable tags (e.g. a biotinylation signal fused to 6xHis tag) gained popularity in recent years, we chose to use a 3xFLAG tag for the following reasons: (i) 3xFLAG tag is a small (22 amino acids) hydrophilic peptide which is likely to be located on the surface of a fusion protein, thus minimizing possible alterations in the structure and function of the target protein; (ii) 3xFLAG sequence contains the binding sites for highly specific and sensitive ANTI-FLAG monoclonal (M2) antibodies that allow isolating highly purified fusion proteins; (iii) one-step purification of target protein includes efficient and gentle competitive elution with synthetic 3xFLAG peptide. As a first step, DNA sequences encoding open reading frames of the PSMB5, PSMA5, and PSMA3 genes were amplified using specific primers from a human cDNA library. The corresponding PCR fragments of PSMB5, PSMA5, and PSMA3 were then cloned into the mammalian expression vector pIRES-hrGFP-1a, which contains a carboxy-terminally located 3xFLAG epitope. The successful recombinant vectors pIRES-hrGFP-1a-PSMB5, pIRES-hrGFP-1aPSMA5, and pIRES-hrGFP-1a-PSMA3 (Fig. 1) were identified by PCR screening and restriction analysis and then were confirmed by sequencing. To overexpress the fusion proteins PSMB5-3xFLAG, PSMA53xFLAG, and PSMA3-3xFLAG human HEK 293T cells were transiently transfected with the corresponding expression vectors. Transfection was carried out using calcium phosphate method as described in the materials and methods section. At 96 h after transfection, whole cell extracts were prepared and analyzed by Western blotting using antibodies against FLAG peptide (Fig. 2). Overexpressed recombinant proteins PSMA5-3xFLAG and PSMA33xFLAG were detected as single bands of expected molecular weights: 29.3 kDa and 31.3 kDa, respectively (Fig. 2B, C). Two distinct bands were detected in whole extracts derived from HEK 293T cells overexpressing fusion protein PSMB5-3xFLAG (Fig. 2A). It has been shown that amino-terminal propeptide (first 59 amino acids) of PSMB5 is removed at the final step of the 20S proteasome assembly resulting in the formation of the catalytically active mature form of PSMB5 [25]. The upper band (30.7 kDa) corresponds to the predicted molecular mass of the full-length PSMB5 protein fused to the 3xFLAG peptide whereas lower band (24.7 kDa) corresponds to the mature forms of PSMB5-3xFLAG (Fig. 2A). This observation indicates that the fusion protein PSMB5-3xFLAG was at least partially incorporated into the cellular proteasome complex after its overexpression in HEK 293T cells. Next, to study the incorporation of overexpressed PSMB53xFLAG, PSMA5-3xFLAG and PSMA3-3xFLAG into macromolecular proteasome complexes we analyzed the distribution of fusion proteins in a glycerol density gradient. Firstly, whole cell extract of control untransfected HEK 293T cells was sedimented in the
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Fig. 1. Schematic representation of the mammalian expression vectors encoding 3xFLAG-tagged 20S proteasome subunits PSMB5, PSMA5, or PSMA3. The human cDNA sequences encoding proteasome subunits PSMB5 (A), PSMA5 (B), or PSMA3 (C) were inserted into the mammalian expression vector pIRES-hrGFP-1a, which contains a carboxy-terminally located 3xFLAG epitope.
Fig. 2. Overexpression of the fusion proteins PSMB5-3xFLAG, PSMA5-3xFLAG and PSMA3-3xFLAG in HEK 293T cells. Human HEK 293T cells were transiently transfected with vectors encoding 3xFLAG-tagged human proteins PSMB5, PSMA5, or PSMA3. Whole cell extracts of non-transfected HEK 293T cells (Control) or HEK 293T cells overexpressing fusion proteins PSMB5-3xFLAG (A), PSMA5-3xFLAG (B), or PSMA3-3xFLAG (C) were analyzed by Western blotting using antibodies against FLAG peptide (Anti-FLAG).
Fig. 3. Distribution of the endogenous and 3xFLAG-tagged proteasome subunits PSMB5, PSMA5, and PSMA3 in soluble extracts of HEK 293T cells. HEK 293T cells were transiently transfected with vectors encoding fusion proteins PSMB5-3xFLAG, PSMA5-3xFLAG, or PSMA3-3xFLAG. 96 h after transfection cells were lysed. Soluble extracts of non-transfected (A) or transfected (B) HEK 293T cells were sedimented in a 5–30% (w/v) glycerol density gradient. The resulting fractions were analyzed by Western blotting using antibodies against the indicated proteasome subunits (A) or antibodies against the FLAG peptide (B).
glycerol density gradient and the distribution of endogenous proteasome subunits PSMB5, PSMA5 or PSMA3 was analyzed by Western blotting of the gradient fractions (Fig. 3A). The 22-kDa cleaved form of PSMB5 was detected in the gradient fractions 7–9 indicating that these fractions correspond to the assembled 20S and/or 26S proteasome complexes. Proteolytically inactive full-length PSMB5 (28 kDa) sedimented significantly slower than its cleaved form and was detected in the gradient fractions 1–4 corresponding to the free form of this protein. Alternatively, these fractions may contain low molecular weight PSMB5 sub-complexes (Fig. 3A, upper panel). Western blotting also showed that the majority of the endogenous proteasome subunit PSMA3 was present in the assembled proteasome complex (Fig. 3A, lower
panel). However, the PSMA5 protein was detected both in the proteasome and low molecular weight fractions (Fig. 3A, middle panel). Next the identical glycerol density gradients were used for the fractionation of whole extracts of HEK 293T cells obtained 96 h after transient transfection with vectors encoding fusion proteins PSMB5-3xFLAG, PSMA5-3xFLAG or PSMA3-3xFLAG. The pattern of distribution of 3xFLAG-tagged proteasome subunits between the gradient fractions (Fig. 3B) was similar to that of the corresponding endogenous proteins (Fig. 3A). These results indicate that each of the overexpressed 3xFLAG-tagged protein was incorporated to a various degree into the cellular proteasome complexes and thus can be used for immunoaffinity purification of 20S proteasomes from human HEK 293T cells.
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For the immunoprecipitation of 20S proteasomes HEK 293T cells were transiently transfected with vectors encoding 3xFLAGtagged proteasome subunits PSMB5, PSMA5 or PSMA3 and then cultured for 96 h. Transfected cells were then lysed under nondenaturing conditions. Whole cell extracts were prepared and subsequently incubated with magnetic beads coated with agarose conjugated with mouse monoclonal antibodies against FLAG peptide (M2). The beads were then intensively washed and the immunoprecipitated proteins bound to beads were eluted with buffer containing 3[FLAG peptide. Eluted proteins were then analyzed by Western blotting using antibodies against FLAG peptides or selected 20S proteasome subunits. As shown in Fig. 4A–C, immunoprecipitates contain overexpressed 3xFLAG-tagged proteins PSMB5, PSMA5 or PSMA3 as well as various endogenous alpha and beta proteasome subunits. Immunoaffinity-purified protein complexes separated by SDS–PAGE were also visualized using SYPRO Ruby (Fig. 4D) or Coomassie Brilliant Blue (Fig. 4E) staining that revealed a set of stained proteins (from 21 to 32 kDa) characteristic of the purified 20S proteasome [12,14]. No immunoprecipitated proteins were detected in the control immunoprecipitates from non-transfected HEK 293T cells (Fig. 4). These data confirmed successful immunoprecipitation of intact 20S proteasome complexes from HEK 293T cells transiently transfected with PSMB53xFLAG, PSMA5-3xFLAG or PSMA3-3xFLAG vectors. Protein bands corresponding to the overexpressed 3xFLAG-tagged subunits had intensities similar to those of untagged endogenous proteasome subunits (Fig. 4D, E), suggesting that the immunoprecipitates did not contain appreciable amounts of of 3xFLAG-tagged proteins in their free form. Colorimetric quantitation of total protein using the BCA protein assay revealed that the total yield of pure 20S
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proteasome from each of three immunoprecipitations was 5 micrograms on average. It is important to note that all steps of the 20S proteasome purification were performed in the absence of ATP which is required for the physical association of the 19S regulatory complex with the 20S core proteasome [26,27]. As shown in Fig. 4A–C, a subunit of the 19S regulatory particle, PSMD6, was not co-immunoprecipitated with fusion proteins PSMB5-3xFLAG, PSMA5-3xFLAG or PSMA3-3xFLAG. Moreover, no protein bands typical for the 19S complex (25–110 kDa) were found after staining with SYPRO Ruby or Coomassie Brilliant Blue (Fig. 4D, E). These data indicate that the 19S regulatory complex was not co-purified with the 20S proteasomes under these experimental conditions. To confirm the functionality of the obtained 20S proteasomes, we tested them for the proteasome-specific peptidase activities. Chymotrypsin- and trypsin-like activities of purified 20S proteasomes were estimated based on the efficiency of hydrolysis of the fluorogenic substrates Suc-LLVY-AMC or Boc-LRR-AMC, respectively [28,29]. The fluorogenic group of these substrates, 7-amino4-methylcoumarin (AMC), increases in fluorescence when released from the peptide by proteolysis. As shown in Fig. 5, all purified 20S proteasomes possessed both chymotrypsin- and trypsin-like activities. Peptidase activities were substantially suppressed when 20S proteasomes were pre-incubated with the specific proteasome inhibitor MG132 (Fig. 5). The specific activities of the affinitypurified 20S proteasomes (Table 1) were measured in the endpoint assay as described in Materials and methods. The level of specific activity of the 20S proteasome obtained according to the purification scheme described in this study is comparable to specific activity of the 20S proteasome obtained by previously
Fig. 4. Immunoprecipitation of intact 20S proteasomes from extracts of HEK 293T cells. HEK 293T cells were transiently transfected with vectors encoding fusion proteins PSMB5-3xFLAG (A, D), PSMA5-3xFLAG (B, E), or PSMA3-3xFLAG (C). 96 h after transfection cells were lysed under non-denaturing conditions and 20S proteasomes were immunoprecipitated using Anti-FLAG M2 magnetic beads. The immunoblots (A-C) display overexpressed 3xFLAG-tagged and various endogenous proteasome subunits in whole cell extract (Input) and in the immunoprecipitate (IP). Affinity-purified 20S proteasomes were separated by SDS–PAGE and stained with SYPRO Ruby (D) or Coomassie Brilliant Blue (E). Non-transfected HEK 293T cells were used as a negative control. Asterisks indicate the positions of the 3xFLAG-tagged subunits (D, E).
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Fig. 5. Affinity-purified 20S proteasomes possess specific peptidase activities. 20S proteasomes were immunoprecipitated (IP) from extracts of HEK 293T cells overexpressing fusion proteins PSMB5-3xFLAG (A), PSMA5-3xFLAG (B), or PSMA3-3xFLAG (C) using Anti-FLAG M2 magnetic beads. Chymotrypsin- and trypsin-like peptidase activities of the affinity-purified 20S proteasomes (1 lg (A and B) or 0.1 lg (C)) were measured based on the efficiency of hydrolysis of the specific fluorogenic substrates Suc-LLVY-AMC and Boc-LRR-AMC, respectively, in the presence or absence of inhibitor MG132. The immunoprecipitated material from non-transfected HEK 293T cells was used as a control (left columns). Values of the background fluorescence (measured in control samples) were taken as 1. Relative activities were calculated as fold induction over the background. Columns represent the mean ± SD from 3 measurements. The p value is calculated using Student’s t-test. ⁄p < 0.001.
Table 1 Specific activities of affinity-purified 20S proteasomes. Overexpressed 3xFLAG-tagged 20S proteasome subunit
PSMB5 PSMA5 PSMA3
Specific activity of the 20S proteasome (pmol hr
1
lg
1 a
)
Chymotrypsin-like
Trypsin-like
7918 ± 84b 7382 ± 47 6223 ± 42
437 ± 13 861 ± 18 /c
a The specific activities of the affinity-purified 20S proteasomes were calculated using AMC-standard curve and presented as the pmol of released AMC per 1 h per 1 lg of proteasome. b Data are presented as mean ± SD from 3 measurements. c Data not available.
published methods. For example, chymotrypsin-like activity of our preparation is 6000–8000 pmol hr 1 lg 1, which is at least twice higher than the same specific activity of 20S proteasome obtained by Tenzer et al. [15]. Thus, we have developed an efficient protocol for rapid immunoaffinity purification of 20S proteasomes from HEK 293T cells transiently overexpressing 3xFLAG-fusion proteasome subunits PSMB5, PSMA5, or PSMA3 using commercially available M2 antibodies. Each of the three proposed IP strategies allows isolating highly purified functional 20S complexes and therefore can be recommended for the routine small-scale purification of the 20S proteasome to be subsequently used in functional studies. Acknowledgments This work was supported by The Program of the Ministry of Education & Science FASI (nos.16.740.11.0366 and 14.740.11.0920) and Russian Foundation for Basic Research (nos. 12-04-31686 mol_a and 13-04-01024). The work was performed using scientific equipment of the center of Shared Usage ’’The Analytical Center of Nano- and Biotechnologies of SPbSPU’’. References [1] A. Hershko, A. Ciechanover, The ubiquitin system, Annu Rev Biochem 67 (1998) 425–479. [2] T. Jung, B. Catalgol, T. Grune, The proteasomal system, Mol Aspects Med 30 (2009) 191–296. [3] K. Tanaka, The proteasome: overview of structure and functions, Proc Jpn Acad Ser B Phys Biol Sci 85 (2009) 12–36. [4] I.M. Konstantinova, A.S. Tsimokha, A.G. Mittenberg, Role of proteasomes in cellular regulation, Int Rev Cell Mol Biol 267 (2008) 59–124.
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Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep
Immunoaffinity purification of the functional 20S proteasome from human cells via transient overexpression of specific proteasome subunits Veronika A. Livinskaya a,b, Nickolai A. Barlev a,c, Andrey A. Nikiforov a,b,⇑ a b c
Institute of Cytology, Russian Academy of Science, Tikhoretsky ave. 4, 194064 Saint Petersburg, Russia Institute of Nanobiotechnologies, Saint Petersburg State Polytechnical University, Polytechnicheskaya 29, 195251 Saint Petersburg, Russia Department of Biochemistry, University of Leicester, Leicester LE1 9HN, United Kingdom
a r t i c l e
i n f o
Article history: Received 15 December 2013 and in revised form 15 February 2014 Available online 28 February 2014 Keywords: Immunoaffinity purification 20S proteasome 3xFLAG peptide PSMB5 PSMA5 PSMA3
a b s t r a c t The proteasome is a multi-subunit proteolytic complex that plays a central role in protein degradation in all eukaryotic cells. It regulates many vital cellular processes therefore its dysfunction can lead to various pathologies including cancer and neurodegeneration. Isolation of enzymatically active proteasomes is a key step to the successful study of the proteasome regulation and functions. Here we describe a simple and efficient protocol for immunoaffinity purification of the functional 20S proteasomes from human HEK 293T cells after transient overexpression of specific proteasome subunits tagged with 3xFLAG. To construct 3xFLAG-fusion proteins, DNA sequences encoding the 20S proteasome subunits PSMB5, PSMA5, and PSMA3 were cloned into mammalian expression vector pIRES-hrGFP-1a. The corresponding recombinant proteins PSMB5-3xFLAG, PSMA5-3xFLAG, or PSMA3-3xFLAG were transiently overexpressed in human HEK 293T cells and were shown to be partially incorporated into the intact proteasome complexes. 20S proteasomes were immunoprecipitated from HEK 293T cell extracts under mild conditions using antibodies against FLAG peptide. Isolation of highly purified 20S proteasomes were confirmed by SDS–PAGE and Western blotting using antibodies against different proteasome subunits. Affinity purified 20S proteasomes were shown to possess chymotrypsin- and trypsin-like peptidase activities confirming their functionality. This simple single-step affinity method of the 20S proteasome purification can be instrumental to subsequent functional studies of proteasomes in human cells. Ó 2014 Elsevier Inc. All rights reserved.
Introduction The ubiquitin–proteasome system (UPS)1 catalyzes the selective degradation of short-lived regulatory proteins and proteins with abnormal conformation in eukaryotic cells. UPS regulates many vital cellular processes such as cell cycle progression, apoptosis, DNA repair, immune responses, protein quality control therefore its ⇑ Corresponding author at: Institute of Cytology, Russian Academy of Science, Tikhoretsky ave. 4, 194064, Saint Petersburg, Russia. Tel.: +7 911 9050330; fax: +7 812 2973541. E-mail address: [email protected] (A.A. Nikiforov). 1 Abbreviations used: UPS, ubiquitin-proteasome system; RP, regulatory particle; HIF-1, hypoxia-inducible factor-1; SRC-3, steroid receptor coactivator-3; HEK, human embryonic kidney; Suc-LLVY-AMC, Succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosine-4methylcoumaryl-7-amide; Boc-LRR-AMC, t-Butyloxycarbonyl-L-leucyl-L-arginyl-Larginine-4-methylcoumaryl-7-amide; AMC, 7-amino-4-methylcoumarin; LB, Luria– Bertani medium; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; HBS, hepes buffered saline; RT, room temperature; PBS, phosphate buffered saline; PMSF, phenylmethylsulfonyl fluoride; WB, washing buffer. http://dx.doi.org/10.1016/j.pep.2014.02.011 1046-5928/Ó 2014 Elsevier Inc. All rights reserved.
dysfunction can lead to various pathologies like cancer or neurodegeneration [1,2]. The 26S proteasome is an ATP-dependent multisubunit protease that degrades polyubiquitinated proteins to small peptides. It is composed of a catalytic 20S core complex, also called the 20S proteasome, and one or two 19S regulatory particles (RP). The 20S proteasome is a 700 kDa barrel-shaped protein complex consisting of four stacked heptameric rings. Each of the two inner rings is composed of seven different b-subunits (PSMB1–7), three of which (PSMB5, PSMB6 and PSMB7) represent chymotrypsin-like, trypsin-like, and caspase-like proteolytic activities of the 20S proteasome. Seven homologous but not identical a-subunits (PSMA1–7) compose each of the two outer rings which regulate substrate access to proteolytic chamber of the 20S proteasome and binding of regulatory complexes. The 19S RP is a 700 kDa protein complex consisting of about 20 various subunits. 19S RP selects substrates for degradation by binding polyubiquitin chains of target proteins which are then deubiquitinated, unfolded and translocated to the proteolytic chamber of the 20S proteasome for breakdown [2–4]. Importantly,
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proteasomes undergo ubiquitinylation by themselves, which affects their proteolytic activities [5]. Besides ubiquitin-dependent protein degradation, 26S proteasome, as well as 20S proteasome alone, also controls ubiquitinindependent cleavage of various regulatory proteins. Tumor suppressor p53, inhibitor of cyclin-dependent kinases p21WAF1/CIP1, transcription factors c-Fos, Fra-1 and HIF-1, calmodulin, steroid receptor coactivator-3 (SRC-3) and many other proteins have been shown to be regulated by ubiquitin-independent proteasomal degradation, mostly via interaction with the PSMA3 subunit of the 20S complex [6–9]. In addition, 20S proteasome catalyzes endoproteolytic cleavage of polypeptide precursors resulting in their maturation [6]. Besides the proteolytic activities 20S proteasomes were shown to possess non-canonical endoribonuclease activity [10,11]. Proteasome purification is a crucial step in the majority of studies focused on proteasome regulation and functions. Conventional proteasome purification usually takes several days and includes three common steps: ultracentrifugation (or gel filtration chromatography), ion exchange chromatography, and hydrophobic interaction chromatography [12–15]. This procedure is performed under strict experimental conditions, such as exposure to high salt concentrations, which can lead to loss of proteasome-associated proteins during purification. Since the last decade, faster and easier affinity-based strategy for proteasome purification has been developed. This approach is based on single-step affinity chromatography after overexpression of epitope-tagged proteasome subunits in yeast or human cells [14,16–18]. Affinity purification of 20S or 26S proteasome is conducted under mild conditions which preserve weak protein–protein interactions. Therefore this method is a powerful tool for purification with further characterization of proteasome-associated proteins [17–20].To date, a substantial body of evidence has been accumulated suggesting that the 20S proteasome exerts important biological functions on its own, independently of the 19S complex [21,22]. Therefore, an implementation of novel robust and simple biochemical approaches to purify functional 20S proteasomes is well justified. Here we present the development of a simple and efficient immunoaffinity method for purification of an enzymatically active 20S proteasomes from cultured human HEK 293T cells via transient overexpression of 3xFLAG-tagged proteasome subunits PSMB5, PSMA5, or PSMA3. Highly purified tagged 20S proteasomes were obtained by immunoprecipitation from cell extracts using monoclonal antibodies to FLAG peptide.
Materials and methods Materials Escherichia coli DH10 cells were used for cloning and plasmid amplification. Human embryonic kidney (HEK) 293T cell line was obtained from the cell culture bank of the Institute of Cytology, RAS (St. Petersburg, Russia). pIRES-hrGFP-1a plasmid was purchased from Agilent Technologies. ANTI-FLAG M2 Magnetic Beads were purchased from Sigma–Aldrich. The following antibodies were used for Western blotting: mouse anti-FLAG (M2), HRP-conjugated goat anti-rabbit and rabbit anti-mouse antibodies (Sigma–Aldrich); rabbit anti-PSMA3, rabbit anti-PSMA5, and rabbit anti-PSMB5 were custom generated in the laboratory [23]; mouse anti-PSMA1, mouse anti-PSMA4, mouse anti-PSMB6, mouse antiPSMB7, and mouse anti-PSMD6 (Enzo Life Sciences). DNA modifying and restriction enzymes were obtained from Fermentas or New England BioLabs. Oligonucleotides were synthesized by Sigma–Aldrich. To measure the peptidase activity of proteasomes the following fluorogenic peptides were used: t-Butyloxycarbonyl-L-leucyl-L-arginyl-L-arginine-4-methylcoumaryl-7-amide
(Boc-LRR-AMC) and Succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosine-4methylcoumaryl-7-amide (Suc-LLVY-AMC) (all purchased from Enzo Life Sciences). Cloning and generation of mammalian expression vectors To generate expression vectors with 3xFLAG-tagged PSMA3, PSMA5 and PSMB5 proteasome subunits in human cells the corresponding DNA sequences (GenBank Accession Nos. NM_002788, NM_002790, and NM_002797, respectively) were amplified from a cDNA library of human K562 cells using the following primers: PSMA3-F 5’-CGTAGGATCCATGAGCTCAATC-3’, PSMA3-R 5’-GTAA CTCGAGCATATTATCATCATC-3’ containing the BamH1 and Xho1 restriction sites, respectively; PSMA5-F 5’-GCCGAATTC AATGTTTCT TACCCGGTC-3’, PSMA5-R 5’-GCGGTCGACGAATGTC CTTGATAACC3’ containing the EcoR1 and SalI restriction sites, respectively; PSMB5-F: 5’-AGTCGAATTCAAT GGCGCTTGCCAGC-3’, PSMB5-R: 5’-GATACTCGAGGGGGGTAGAGCCAC-3’ containing the EcoR1 and Xho1 restriction sites, respectively. Amplified fragments of PSMA3, PSMA5 and PSMB5 were then inserted into the plasmid pIRES-hrGFP-1a digested with the matching pairs of restriction enzymes. Competent E. coli DH10 cells were transformed with obtained recombinant plasmids pIRES-hrGFP-1a-PSMA3, pIRES-hrGFP-1aPSMA5, or pIRES-hrGFP-1a-PSMB5 and plated onto LB agar plates supplemented with ampicillin (100 lg/ml). Transformants were screened by PCR and were subsequently verified by the restriction analysis. The accuracy of cloning was confirmed by sequencing. Cell culture and transfection HEK 293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS (Gibco), 2 mM L-glutamine and 20 lg/ml gentamicin. The cells were cultured at 37 °C in a humidified atmosphere of 5% CO2. Transient transfection of HEK 293T cells with vectors pIRES-hrGFP-1a-PSMA3, pIRES-hrGFP-1a-PSMA5, or pIRES-hrGFP1a-PSMB5 was done using the calcium phosphate method. Cells were seeded in a 100 mm culture plate and then grown to 50–70% confluence. One hour before transfection fresh medium (10 ml) was added to cells. Pure plasmid DNA (8–10 lg) was dissolved in 600 ll of 250 mM CaCl2 solution. Then the mixture was carefully added to 600 ll of 2 HBS buffer (50 mM Hepes, 10 mM KCl, 1.5 mM Na2HPO4, and 280 mM NaCl, pH 7.05) with concomitant vortexing. After 20 min incubation at room temperature (RT) the mixture (final volume of 1200 ll) was added dropwise onto the culture plate. The culture plate was carefully shaken to equally disperse the calcium phosphate precipitates. Cells were incubated for 24–96 h at 37 °C, and then the whole cell extract was prepared and used for immunoprecipitation or Western blotting analysis. Immunoaffinity purification of 20S proteasome HEK 293T cells, grown in a four 100 mm culture plates (5x10E6 cells per plate), were transfected with vectors pIRES-hrGFP-1aPSMB5, pIRES-hrGFP-1a-PSMA5, or pIRES-hrGFP-1a-PSMA3 encoding fusion proteins PSMB5-3xFLAG, PSMA5-3xFLAG, or PSMA33xFLAG, respectively. 24 h after transfection cells from four 100 mm plates (for each transfection) were plated onto six 150 mm plates and incubated for 72 h at 37 °C. 96 h after transfection cells were washed twice with ice-cold PBS and lysed in the lysis buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 0.3% Triton X-100, and 0.5 mM PMSF) by agitation for 40 min at 4 °C. The resulting cell lysate was clarified by centrifugation at 20,000g for 30 min at 4 °C, and the supernatant was collected. The cell extract
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was incubated with 100 ll of Anti-FLAG M2 magnetic beads overnight at 4 °C under gentle rocking. The beads were then washed five times with 5 ml of washing buffer (WB) containing 50 mM Tris (pH 7.5), 150 mM NaCl, and 0.5 mM PMSF. Immunoprecipitated proteins were eluted at 4 °C with 500 ll of WB supplemented with 3[FLAG peptide (300 lg/ml). 3[FLAG peptide was then removed by serial concentration on 10 kDa concentrators (Millipore). The immunoaffinity-purified protein complexes were characterized by SDS–PAGE, Western blotting and proteasome activity, as described below. Protein determination, SDS–PAGE, and western blotting Protein concentration of samples was determined using BCA protein assay kit (Pierce) according to the manufacturer’s protocol. All samples were prepared by boiling in a Laemmli buffer (62.5 mM Tris–HCl (pH 6.8), 2% SDS, 0.05% bromophenol blue, 10% glycerol, and 100 mM DTT) for 7 min. Then proteins were resolved by 12% SDS–polyacrylamide gel electrophoresis (SDS–PAGE) followed by visualization using Coomassie blue staining. For Western blotting, proteins were transferred onto a PVDF membrane after separation by SDS–PAGE. The obtained membrane was blocked with PBS supplemented with 5% (w/v) non-fat dry milk and 0.1% (v/v) Tween 20 for 1 h at RT or overnight at 4 °C. Following blocking, the membrane was incubated with appropriate primary and HRP-conjugated secondary antibodies was performed in PBS supplemented with 1% (w/v) non-fat dry milk and 0.1% (v/v) Tween 20 for 1 h at RT. The membranes were washed six times in PBS supplemented with 0.1% (v/v) Tween 20 after incubations with primary and secondary antibodies. ECL detection was performed with SuperSignal system (Thermo Fisher Scientific) according to the manufacturer’s protocol. Proteasome activity assay The chymotrypsin- and trypsin-like peptidase activities of purified 20S proteasomes were calculated based on the efficiency of hydrolysis of the respective fluorogenic substrates Suc-LLVY-AMC and Boc-LRR-AMC. The assay was conducted in the buffer containing 150 lM Suc-LLVY-AMC or 480 lM Boc-LRR-AMC, 50 mM Tris–HCl (pH 7.5), 5 mM MgCl2, 40 mM KCl, and 1 mM DTT at 37 °C for 1 h. The liberated 7-amino-4-methylcoumarin (AMC) was measured fluorimetrically (excitation at 365 nm, emission at 440 nm). For the control of specificity 20S proteasomes were treated with a proteasome inhibitor MG132 or mock-treated with DMSO for 20 min at RT before adding the substrates. The specific activity of the purified 20S proteasome was calculated using AMC-standard curve and presented as the pmol of released AMC per 1 h per 1 lg of proteasome. Glycerol density gradient centrifugation HEK 293T cells were transfected with vectors pIRES-hrGFP-1aPSMB5, pIRES-hrGFP-1a-PSMA5, or pIRES-hrGFP-1a-PSMA3. 96 h after transfection cells were washed twice with ice-cold PBS and lysed in the lysis buffer (50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 0.1% Triton X-100, and 0.5 mM PMSF) by agitation for 40 min at 4 °C. The resulting cell lysates were clarified by centrifugation at 25,000g for 30 min at 4 °C, and the supernatants were collected. Soluble extracts from control and transfected cells were loaded onto a 5 to 30% glycerol density gradient, followed by centrifugation at 80,000 g for 17 h at 4 °C. Eleven fractions were collected from the top of the gradients, and the proteins were precipitated with 80% ethanol. Then precipitated proteins were dissolved in
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Laemmli buffer, subjected to SDS–PAGE and analyzed by Western blotting.
Results and discussion The fusion of a protein of interest with affinity tag is a commonly used method for purification of recombinant proteins. Immunoaffinity purification of fusion proteins using antibodies against tag peptide is performed under mild non-denaturing conditions that preserve physiological protein–protein associations and thus can be used for isolation of native protein complexes from mammalian cells [24]. This study aimed to establish a simple and efficient method for immunoaffinity purification of functional 20S proteasomes from human HEK 293T cells after transient overexpression of 3xFLAG-tagged proteasome subunits using antibodies to FLAG peptide. Although TEV protease cleavable tags (e.g. a biotinylation signal fused to 6xHis tag) gained popularity in recent years, we chose to use a 3xFLAG tag for the following reasons: (i) 3xFLAG tag is a small (22 amino acids) hydrophilic peptide which is likely to be located on the surface of a fusion protein, thus minimizing possible alterations in the structure and function of the target protein; (ii) 3xFLAG sequence contains the binding sites for highly specific and sensitive ANTI-FLAG monoclonal (M2) antibodies that allow isolating highly purified fusion proteins; (iii) one-step purification of target protein includes efficient and gentle competitive elution with synthetic 3xFLAG peptide. As a first step, DNA sequences encoding open reading frames of the PSMB5, PSMA5, and PSMA3 genes were amplified using specific primers from a human cDNA library. The corresponding PCR fragments of PSMB5, PSMA5, and PSMA3 were then cloned into the mammalian expression vector pIRES-hrGFP-1a, which contains a carboxy-terminally located 3xFLAG epitope. The successful recombinant vectors pIRES-hrGFP-1a-PSMB5, pIRES-hrGFP-1aPSMA5, and pIRES-hrGFP-1a-PSMA3 (Fig. 1) were identified by PCR screening and restriction analysis and then were confirmed by sequencing. To overexpress the fusion proteins PSMB5-3xFLAG, PSMA53xFLAG, and PSMA3-3xFLAG human HEK 293T cells were transiently transfected with the corresponding expression vectors. Transfection was carried out using calcium phosphate method as described in the materials and methods section. At 96 h after transfection, whole cell extracts were prepared and analyzed by Western blotting using antibodies against FLAG peptide (Fig. 2). Overexpressed recombinant proteins PSMA5-3xFLAG and PSMA33xFLAG were detected as single bands of expected molecular weights: 29.3 kDa and 31.3 kDa, respectively (Fig. 2B, C). Two distinct bands were detected in whole extracts derived from HEK 293T cells overexpressing fusion protein PSMB5-3xFLAG (Fig. 2A). It has been shown that amino-terminal propeptide (first 59 amino acids) of PSMB5 is removed at the final step of the 20S proteasome assembly resulting in the formation of the catalytically active mature form of PSMB5 [25]. The upper band (30.7 kDa) corresponds to the predicted molecular mass of the full-length PSMB5 protein fused to the 3xFLAG peptide whereas lower band (24.7 kDa) corresponds to the mature forms of PSMB5-3xFLAG (Fig. 2A). This observation indicates that the fusion protein PSMB5-3xFLAG was at least partially incorporated into the cellular proteasome complex after its overexpression in HEK 293T cells. Next, to study the incorporation of overexpressed PSMB53xFLAG, PSMA5-3xFLAG and PSMA3-3xFLAG into macromolecular proteasome complexes we analyzed the distribution of fusion proteins in a glycerol density gradient. Firstly, whole cell extract of control untransfected HEK 293T cells was sedimented in the
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Fig. 1. Schematic representation of the mammalian expression vectors encoding 3xFLAG-tagged 20S proteasome subunits PSMB5, PSMA5, or PSMA3. The human cDNA sequences encoding proteasome subunits PSMB5 (A), PSMA5 (B), or PSMA3 (C) were inserted into the mammalian expression vector pIRES-hrGFP-1a, which contains a carboxy-terminally located 3xFLAG epitope.
Fig. 2. Overexpression of the fusion proteins PSMB5-3xFLAG, PSMA5-3xFLAG and PSMA3-3xFLAG in HEK 293T cells. Human HEK 293T cells were transiently transfected with vectors encoding 3xFLAG-tagged human proteins PSMB5, PSMA5, or PSMA3. Whole cell extracts of non-transfected HEK 293T cells (Control) or HEK 293T cells overexpressing fusion proteins PSMB5-3xFLAG (A), PSMA5-3xFLAG (B), or PSMA3-3xFLAG (C) were analyzed by Western blotting using antibodies against FLAG peptide (Anti-FLAG).
Fig. 3. Distribution of the endogenous and 3xFLAG-tagged proteasome subunits PSMB5, PSMA5, and PSMA3 in soluble extracts of HEK 293T cells. HEK 293T cells were transiently transfected with vectors encoding fusion proteins PSMB5-3xFLAG, PSMA5-3xFLAG, or PSMA3-3xFLAG. 96 h after transfection cells were lysed. Soluble extracts of non-transfected (A) or transfected (B) HEK 293T cells were sedimented in a 5–30% (w/v) glycerol density gradient. The resulting fractions were analyzed by Western blotting using antibodies against the indicated proteasome subunits (A) or antibodies against the FLAG peptide (B).
glycerol density gradient and the distribution of endogenous proteasome subunits PSMB5, PSMA5 or PSMA3 was analyzed by Western blotting of the gradient fractions (Fig. 3A). The 22-kDa cleaved form of PSMB5 was detected in the gradient fractions 7–9 indicating that these fractions correspond to the assembled 20S and/or 26S proteasome complexes. Proteolytically inactive full-length PSMB5 (28 kDa) sedimented significantly slower than its cleaved form and was detected in the gradient fractions 1–4 corresponding to the free form of this protein. Alternatively, these fractions may contain low molecular weight PSMB5 sub-complexes (Fig. 3A, upper panel). Western blotting also showed that the majority of the endogenous proteasome subunit PSMA3 was present in the assembled proteasome complex (Fig. 3A, lower
panel). However, the PSMA5 protein was detected both in the proteasome and low molecular weight fractions (Fig. 3A, middle panel). Next the identical glycerol density gradients were used for the fractionation of whole extracts of HEK 293T cells obtained 96 h after transient transfection with vectors encoding fusion proteins PSMB5-3xFLAG, PSMA5-3xFLAG or PSMA3-3xFLAG. The pattern of distribution of 3xFLAG-tagged proteasome subunits between the gradient fractions (Fig. 3B) was similar to that of the corresponding endogenous proteins (Fig. 3A). These results indicate that each of the overexpressed 3xFLAG-tagged protein was incorporated to a various degree into the cellular proteasome complexes and thus can be used for immunoaffinity purification of 20S proteasomes from human HEK 293T cells.
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For the immunoprecipitation of 20S proteasomes HEK 293T cells were transiently transfected with vectors encoding 3xFLAGtagged proteasome subunits PSMB5, PSMA5 or PSMA3 and then cultured for 96 h. Transfected cells were then lysed under nondenaturing conditions. Whole cell extracts were prepared and subsequently incubated with magnetic beads coated with agarose conjugated with mouse monoclonal antibodies against FLAG peptide (M2). The beads were then intensively washed and the immunoprecipitated proteins bound to beads were eluted with buffer containing 3[FLAG peptide. Eluted proteins were then analyzed by Western blotting using antibodies against FLAG peptides or selected 20S proteasome subunits. As shown in Fig. 4A–C, immunoprecipitates contain overexpressed 3xFLAG-tagged proteins PSMB5, PSMA5 or PSMA3 as well as various endogenous alpha and beta proteasome subunits. Immunoaffinity-purified protein complexes separated by SDS–PAGE were also visualized using SYPRO Ruby (Fig. 4D) or Coomassie Brilliant Blue (Fig. 4E) staining that revealed a set of stained proteins (from 21 to 32 kDa) characteristic of the purified 20S proteasome [12,14]. No immunoprecipitated proteins were detected in the control immunoprecipitates from non-transfected HEK 293T cells (Fig. 4). These data confirmed successful immunoprecipitation of intact 20S proteasome complexes from HEK 293T cells transiently transfected with PSMB53xFLAG, PSMA5-3xFLAG or PSMA3-3xFLAG vectors. Protein bands corresponding to the overexpressed 3xFLAG-tagged subunits had intensities similar to those of untagged endogenous proteasome subunits (Fig. 4D, E), suggesting that the immunoprecipitates did not contain appreciable amounts of of 3xFLAG-tagged proteins in their free form. Colorimetric quantitation of total protein using the BCA protein assay revealed that the total yield of pure 20S
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proteasome from each of three immunoprecipitations was 5 micrograms on average. It is important to note that all steps of the 20S proteasome purification were performed in the absence of ATP which is required for the physical association of the 19S regulatory complex with the 20S core proteasome [26,27]. As shown in Fig. 4A–C, a subunit of the 19S regulatory particle, PSMD6, was not co-immunoprecipitated with fusion proteins PSMB5-3xFLAG, PSMA5-3xFLAG or PSMA3-3xFLAG. Moreover, no protein bands typical for the 19S complex (25–110 kDa) were found after staining with SYPRO Ruby or Coomassie Brilliant Blue (Fig. 4D, E). These data indicate that the 19S regulatory complex was not co-purified with the 20S proteasomes under these experimental conditions. To confirm the functionality of the obtained 20S proteasomes, we tested them for the proteasome-specific peptidase activities. Chymotrypsin- and trypsin-like activities of purified 20S proteasomes were estimated based on the efficiency of hydrolysis of the fluorogenic substrates Suc-LLVY-AMC or Boc-LRR-AMC, respectively [28,29]. The fluorogenic group of these substrates, 7-amino4-methylcoumarin (AMC), increases in fluorescence when released from the peptide by proteolysis. As shown in Fig. 5, all purified 20S proteasomes possessed both chymotrypsin- and trypsin-like activities. Peptidase activities were substantially suppressed when 20S proteasomes were pre-incubated with the specific proteasome inhibitor MG132 (Fig. 5). The specific activities of the affinitypurified 20S proteasomes (Table 1) were measured in the endpoint assay as described in Materials and methods. The level of specific activity of the 20S proteasome obtained according to the purification scheme described in this study is comparable to specific activity of the 20S proteasome obtained by previously
Fig. 4. Immunoprecipitation of intact 20S proteasomes from extracts of HEK 293T cells. HEK 293T cells were transiently transfected with vectors encoding fusion proteins PSMB5-3xFLAG (A, D), PSMA5-3xFLAG (B, E), or PSMA3-3xFLAG (C). 96 h after transfection cells were lysed under non-denaturing conditions and 20S proteasomes were immunoprecipitated using Anti-FLAG M2 magnetic beads. The immunoblots (A-C) display overexpressed 3xFLAG-tagged and various endogenous proteasome subunits in whole cell extract (Input) and in the immunoprecipitate (IP). Affinity-purified 20S proteasomes were separated by SDS–PAGE and stained with SYPRO Ruby (D) or Coomassie Brilliant Blue (E). Non-transfected HEK 293T cells were used as a negative control. Asterisks indicate the positions of the 3xFLAG-tagged subunits (D, E).
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V.A. Livinskaya et al. / Protein Expression and Purification 97 (2014) 37–43
Fig. 5. Affinity-purified 20S proteasomes possess specific peptidase activities. 20S proteasomes were immunoprecipitated (IP) from extracts of HEK 293T cells overexpressing fusion proteins PSMB5-3xFLAG (A), PSMA5-3xFLAG (B), or PSMA3-3xFLAG (C) using Anti-FLAG M2 magnetic beads. Chymotrypsin- and trypsin-like peptidase activities of the affinity-purified 20S proteasomes (1 lg (A and B) or 0.1 lg (C)) were measured based on the efficiency of hydrolysis of the specific fluorogenic substrates Suc-LLVY-AMC and Boc-LRR-AMC, respectively, in the presence or absence of inhibitor MG132. The immunoprecipitated material from non-transfected HEK 293T cells was used as a control (left columns). Values of the background fluorescence (measured in control samples) were taken as 1. Relative activities were calculated as fold induction over the background. Columns represent the mean ± SD from 3 measurements. The p value is calculated using Student’s t-test. ⁄p < 0.001.
Table 1 Specific activities of affinity-purified 20S proteasomes. Overexpressed 3xFLAG-tagged 20S proteasome subunit
PSMB5 PSMA5 PSMA3
Specific activity of the 20S proteasome (pmol hr
1
lg
1 a
)
Chymotrypsin-like
Trypsin-like
7918 ± 84b 7382 ± 47 6223 ± 42
437 ± 13 861 ± 18 /c
a The specific activities of the affinity-purified 20S proteasomes were calculated using AMC-standard curve and presented as the pmol of released AMC per 1 h per 1 lg of proteasome. b Data are presented as mean ± SD from 3 measurements. c Data not available.
published methods. For example, chymotrypsin-like activity of our preparation is 6000–8000 pmol hr 1 lg 1, which is at least twice higher than the same specific activity of 20S proteasome obtained by Tenzer et al. [15]. Thus, we have developed an efficient protocol for rapid immunoaffinity purification of 20S proteasomes from HEK 293T cells transiently overexpressing 3xFLAG-fusion proteasome subunits PSMB5, PSMA5, or PSMA3 using commercially available M2 antibodies. Each of the three proposed IP strategies allows isolating highly purified functional 20S complexes and therefore can be recommended for the routine small-scale purification of the 20S proteasome to be subsequently used in functional studies. Acknowledgments This work was supported by The Program of the Ministry of Education & Science FASI (nos.16.740.11.0366 and 14.740.11.0920) and Russian Foundation for Basic Research (nos. 12-04-31686 mol_a and 13-04-01024). The work was performed using scientific equipment of the center of Shared Usage ’’The Analytical Center of Nano- and Biotechnologies of SPbSPU’’. References [1] A. Hershko, A. Ciechanover, The ubiquitin system, Annu Rev Biochem 67 (1998) 425–479. [2] T. Jung, B. Catalgol, T. Grune, The proteasomal system, Mol Aspects Med 30 (2009) 191–296. [3] K. Tanaka, The proteasome: overview of structure and functions, Proc Jpn Acad Ser B Phys Biol Sci 85 (2009) 12–36. [4] I.M. Konstantinova, A.S. Tsimokha, A.G. Mittenberg, Role of proteasomes in cellular regulation, Int Rev Cell Mol Biol 267 (2008) 59–124.
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