A Tandem Affinity Tag for Two-step Purification under Fully Denaturing Conditions
2006; Elsevier BV; Volume: 5; Issue: 4 Linguagem: Inglês
10.1074/mcp.m500368-mcp200
ISSN1535-9484
AutoresChristian Tagwerker, Karin Flick, Meng Cui, Cortnie Guerrero, Yimeng Dou, Bernhard Auer, Pierre Baldi, Lan Huang, Peter Kaiser,
Tópico(s)Biotin and Related Studies
ResumoTandem affinity strategies reach exceptional protein purification grades and have considerably improved the outcome of mass spectrometry-based proteomic experiments. However, current tandem affinity tags are incompatible with two-step purification under fully denaturing conditions. Such stringent purification conditions are desirable for mass spectrometric analyses of protein modifications as they result in maximal preservation of posttranslational modifications. Here we describe the histidine-biotin (HB) tag, a new tandem affinity tag for two-step purification under denaturing conditions. The HB tag consists of a hexahistidine tag and a bacterially derived in vivo biotinylation signal peptide that induces efficient biotin attachment to the HB tag in yeast and mammalian cells. HB-tagged proteins can be sequentially purified under fully denaturing conditions, such as 8 m urea, by Ni2+ chelate chromatography and binding to streptavidin resins. The stringent separation conditions compatible with the HB tag prevent loss of protein modifications, and the high purification grade achieved by the tandem affinity strategy facilitates mass spectrometric analysis of posttranslational modifications. Ubiquitination is a particularly sensitive protein modification that is rapidly lost during purification under native conditions due to ubiquitin hydrolase activity. The HB tag is ideal to study ubiquitination because the denaturing conditions inhibit hydrolase activity, and the tandem affinity strategy greatly reduces nonspecific background. We tested the HB tag in proteome-wide ubiquitin profiling experiments in yeast and identified a number of known ubiquitinated proteins as well as so far unidentified candidate ubiquitination targets. In addition, the stringent purification conditions compatible with the HB tag allow effective mass spectrometric identification of in vivo cross-linked protein complexes, thereby expanding proteomic analyses to the description of weakly or transiently associated protein complexes. Tandem affinity strategies reach exceptional protein purification grades and have considerably improved the outcome of mass spectrometry-based proteomic experiments. However, current tandem affinity tags are incompatible with two-step purification under fully denaturing conditions. Such stringent purification conditions are desirable for mass spectrometric analyses of protein modifications as they result in maximal preservation of posttranslational modifications. Here we describe the histidine-biotin (HB) tag, a new tandem affinity tag for two-step purification under denaturing conditions. The HB tag consists of a hexahistidine tag and a bacterially derived in vivo biotinylation signal peptide that induces efficient biotin attachment to the HB tag in yeast and mammalian cells. HB-tagged proteins can be sequentially purified under fully denaturing conditions, such as 8 m urea, by Ni2+ chelate chromatography and binding to streptavidin resins. The stringent separation conditions compatible with the HB tag prevent loss of protein modifications, and the high purification grade achieved by the tandem affinity strategy facilitates mass spectrometric analysis of posttranslational modifications. Ubiquitination is a particularly sensitive protein modification that is rapidly lost during purification under native conditions due to ubiquitin hydrolase activity. The HB tag is ideal to study ubiquitination because the denaturing conditions inhibit hydrolase activity, and the tandem affinity strategy greatly reduces nonspecific background. We tested the HB tag in proteome-wide ubiquitin profiling experiments in yeast and identified a number of known ubiquitinated proteins as well as so far unidentified candidate ubiquitination targets. In addition, the stringent purification conditions compatible with the HB tag allow effective mass spectrometric identification of in vivo cross-linked protein complexes, thereby expanding proteomic analyses to the description of weakly or transiently associated protein complexes. Mass spectrometric analysis of proteins has tremendously contributed to our understanding of biological systems. Mapping of covalent protein modifications by mass spectrometric approaches has made it possible to identify and rapidly evaluate the biological significance of modifications. In addition, identification of protein complexes by mass spectrometry has allowed investigators to connect cellular pathways and to describe the dynamics of protein complexes (1Aebersold R. Mann M. Mass spectrometry-based proteomics.Nature. 2003; 422: 198-207Crossref PubMed Scopus (5484) Google Scholar, 2Yates III, J.R. Mass spectral analysis in proteomics.Annu. Rev. Biophys. Biomol. Struct. 2004; 33: 297-316Crossref PubMed Scopus (249) Google Scholar). These approaches typically require a high degree of purification of proteins or protein complexes. Importantly to get a genuine picture of the in vivo situation it is essential to avoid any changes in protein modification or protein complex composition that might occur during the purification procedure. Two-step purification strategies have been proven to be very effective in reducing nonspecific background, which is particularly important for the analyses of complex protein samples (3Puig O. Caspary F. Rigaut G. Rutz B. Bouveret E. Bragado-Nilsson E. Wilm M. Seraphin B. The tandem affinity purification (TAP) method: a general procedure of protein complex purification.Methods. 2001; 24: 218-229Crossref PubMed Scopus (1407) Google Scholar). The first widely and successfully used tandem affinity tag was the TAP 1The abbreviations used are: TAP, tandem affinity purification; CBP, calmodulin-binding peptide; HB, histidine-biotin; HBH, histidine-biotin-histidine; HBT, histidine-biotin-TEV; 1-D, one-dimensional; SCX, strong cation exchange; GO, Gene Ontology; TEV, tobacco etch virus; HRP, horseradish peroxidase; SUMO, small ubiquitin-like modifier. 1The abbreviations used are: TAP, tandem affinity purification; CBP, calmodulin-binding peptide; HB, histidine-biotin; HBH, histidine-biotin-histidine; HBT, histidine-biotin-TEV; 1-D, one-dimensional; SCX, strong cation exchange; GO, Gene Ontology; TEV, tobacco etch virus; HRP, horseradish peroxidase; SUMO, small ubiquitin-like modifier. tag, which consists of the immunoglobulin-interacting domain of Protein A and a calmodulin-binding peptide (CBP) and allows sequential purification based on Protein A/IgG-agarose and CBP/calmodulin-bead affinities (4Rigaut G. Shevchenko A. Rutz B. Wilm M. Mann M. Seraphin B. A generic protein purification method for protein complex characterization and proteome exploration.Nat. Biotechnol. 1999; 17: 1030-1032Crossref PubMed Scopus (2268) Google Scholar). Other tandem affinity tags include a modified version of the TAP tag in which the CBP part is replaced by the S-tag (5Cheeseman I.M. Brew C. Wolyniak M. Desai A. Anderson S. Muster N. Yates J.R. Huffaker T.C. Drubin D.G. Barnes G. Implication of a novel multiprotein Dam1p complex in outer kinetochore function.J. Cell Biol. 2001; 155: 1137-1145Crossref PubMed Scopus (146) Google Scholar) and combinations of multihistidine tags with FLAG (6Hannich J.T. Lewis A. Kroetz M.B. Li S.J. Heide H. Emili A. Hochstrasser M. Defining the SUMO-modified proteome by multiple approaches in Saccharomyces cerevisiae.J. Biol. Chem. 2005; 280: 4102-4110Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar, 7Denison C. Rudner A.D. Gerber S.A. Bakalarski C.E. Moazed D. Gygi S.P. A proteomic strategy for gaining insights into protein sumoylation in yeast.Mol. Cell. Proteomics. 2004; 4: 246-254Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar) or Myc tags (8Graumann J. Dunipace L.A. Seol J.H. McDonald W.H. Yates III, J.R. Wold B.J. Deshaies R.J. Applicability of tandem affinity purification MudPIT to pathway proteomics in yeast.Mol. Cell. Proteomics. 2004; 3: 226-237Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). The available tandem affinity purification strategies require native conditions in at least one of the purification steps and are therefore susceptible to loss of posttranslational modifications during cell lysis and purification because modifying as well as demodifying enzymes remain active under these conditions. To overcome these limitations we developed a tandem affinity tag, the HB tag, that allows two-step purification under fully denaturing conditions. Yeast strains used in this study are all isogenic to 15DaubΔ, bar1Δ ura3Δns, a derivative of BF264-15D (9Reed S.I. Hadwiger J.A. Lorincz A.T. Protein kinase activity associated with the product of the yeast cell division cycle gene CDC28.Proc. Natl. Acad. Sci. U S A. 1985; 82: 4055-4059Crossref PubMed Scopus (217) Google Scholar). Strains were grown in standard culture medium, and standard yeast genetic methods were used (10Guthrie C. Fink G.R. Guide to Yeast Genetics and Molecular Biology. 194. Academic Press, Inc., San Diego, CA1991Google Scholar). Carboxyl-terminal tagging of Skp1 and Met4 was performed by a PCR-based method as described previously (11Longtine M.S. McKenzie III, A. Demarini D.J. Shah N.G. Wach A. Brachat A. Philippsen P. Pringle J.R. Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae.Yeast. 1998; 14: 953-961Crossref PubMed Scopus (4086) Google Scholar). For this purpose the HBH tag was inserted into the pFA6a-kanMX6 plasmid to generate a PCR template for the PCR-based HBH tagging (12Bahler J. Wu J.Q. Longtine M.S. Shah N.G. McKenzie III, A. Steever A.B. Wach A. Philippsen P. Pringle J.R. Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe.Yeast. 1998; 14: 943-951Crossref PubMed Scopus (1717) Google Scholar). Similar PCR tagging templates were constructed with other HB tag modules described in Fig. 1A. A BamHI fragment containing HBT-ubiquitin was inserted into the yeast expression vector YEpURA-CUP, which contains the inducible CUP1 promoter (region −463 to +1 of the CUP1 gene) and the termination sequence from the CYC1 gene (region +328 to +552) in the YEplac195 vector backbone (13Gietz R.D. Sugino A. New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites.Gene (Amst.). 1988; 74: 527-534Crossref PubMed Scopus (2500) Google Scholar). To express HBT-ubiquitin in mammalian cells a BamHI fragment containing the HBT tag fused to ubiquitin was inserted into pcDNA3.1 (Invitrogen). Cells expressing either carboxyl-terminal HBH-tagged Met4 (strain PY1226) or untagged Met4 (PY236) were grown in 300 ml of YEPD (1% yeast extract, 2% peptone, 2% dextrose) to A600 = 1.5, and cells were harvested by filtration, washed in ice-cold water, and frozen in 12 tubes at −80 °C. Cells were lysed with glass beads in 400 μl of buffer-1 (8 m urea, 300 mm NaCl, 0.5% Nonidet P-40, 50 mm sodium phosphate, 50 mm Tris, pH 8, 1 mm PMSF, 2 mg/ml aprotinin, leupeptin, and pepstatin A) per tube in a FastPrep FP120 system (setting 4.5) for 80 s (Qbiogene, Carlsbad, CA). After removal of the glass beads an additional 400 μl of buffer-1 was added to each tube. Lysates were centrifuged for 15 min at 15,700 × g. Cleared lysates were pooled and incubated with Ni2+-Sepharose (750-μl slurry preequilibrated in buffer-1) (Amersham Biosciences) for 6 h at room temperature. Ni2+-Sepharose was then washed in 5 ml of buffer-1; 5 ml of buffer-1, pH 6.3; and 5 ml of buffer-1, pH 6.3, + 10 mm imidazole. Proteins were eluted in 4 ml of buffer-2 (8 m urea, 200 mm NaCl, 50 mm sodium phosphate, 2% SDS, 10 mm EDTA, 100 mm Tris, pH 4.3). The pH of the eluate was adjusted to pH 8.0, and then the eluate was loaded onto immobilized streptavidin (400-μl slurry preequilibrated in buffer-3 (8 m urea, 200 mm NaCl, 0.2% SDS, 100 mm Tris, pH 8.0)) (Pierce). After incubation overnight at room temperature the streptavidin beads were washed in 3 × 1 ml of buffer-3, 3 × 1 ml of buffer-3 with 2% SDS, and then 3 × 1 ml of buffer-3 without SDS. Streptavidin beads were kept in buffer-3 without SDS at 4 °C until further preparations. Cells carrying either HBH-tagged (PY1259) or untagged Skp1 (PY236) were grown in 300 ml of YEPD to A600 = 1.8. Formaldehyde was then added to a final concentration of 1%, and cells were incubated at 30 °C for 10 min. Cross-linking was quenched by the addition of 125 mm glycine for 5 min at 30 °C. Cells were harvested by filtration, washed in ice-cold water, and stored in six tubes at −80 °C. Cell lysis and purification were essentially done as described for Met4HBH with the following exceptions. Cells were lysed in 500 μl of buffer-1/tube for 3 × 40 s in a FastPrep system. After removal of the glass beads 500 μl of buffer-1 was added. Yeast cells expressing HBT-ubiquitin or untagged ubiquitin under control of the CUP1 promoter from a 2μ plasmid were grown in a total of 200 ml of synthetic complete medium lacking uracil (SC-URA medium) (10Guthrie C. Fink G.R. Guide to Yeast Genetics and Molecular Biology. 194. Academic Press, Inc., San Diego, CA1991Google Scholar) supplemented with 4 μm biotin at 30 °C to A600 ∼0.8. Expression of untagged or HBT-ubiquitin was induced for 3 h by addition of CuSO4 (100 μm final concentration). Buffers containing 8 m urea were prepared fresh and incubated for 1 h at room temperature with 1% ion exchange resin (Amberlite IRN-150L, Amersham Biosciences) prior to adding other buffer ingredients to remove isocyanate impurities, which can lead to carbamylation of lysine residues and interfere with protein analyses. Cells were lysed with glass beads for 4 × 40 s (setting 4.5) in a FastPrep FP120 system (Qbiogene) in buffer A (8 m urea, 300 mm NaCl, 0.5% Nonidet P-40, 50 mm NaH2PO4) + 1 mm PMSF. Cell debris were removed by centrifugation for 10 min at 16,100 × g. The whole cell lysate was incubated with Ni2+-Sepharose 6 FastFlow resin (Amersham Biosciences) for 2 h at room temperature (50 μl of a 50% slurry for each milligram of total protein lysate). The resin was washed in column format sequentially with 10 bed volumes each of buffer-A, pH 8; 10 bed volumes of buffer A, pH 6.3; and buffer A, pH 6.3, containing 20 mm imidazole. PMSF was omitted in wash buffers. HB-tagged proteins were eluted in 5 bed volumes of buffer B (8 m urea, 200 mm NaCl, 50 mm NaH2PO4, 2% SDS, 10 mm EDTA, 100 mm Tris, pH 4.3). The eluates were adjusted to pH 8 prior to incubation with ImmunoPure immobilized streptavidin-agarose (Pierce) overnight at room temperature (10 μl of a 50% slurry for each milligram of total protein lysate used in the first purification step). The streptavidin resin was washed sequentially with 25 bed volumes of each of the following: buffer C (8 m urea, 200 mm NaCl, 2% SDS, 100 mm Tris, pH 8), buffer D (8 m urea, 1.2 m NaCl, 0.2% SDS, 100 mm Tris, 10% ethanol, 10% isopropanol, pH 8), buffer E (8 m urea, 200 mm NaCl, 0.2% SDS, 100 mm Tris, 10% ethanol, 10% isopropanol, pH 5 and 9), and buffer F (8 m urea, 100 mm Tris, pH 8). For on-bead Lys-C/trypsin digestion, endoproteinase Lys-C (Wako) was directly added after reducing the 8 m urea buffer volume to the volume of remaining beads, and samples were incubated for 4 h at 37 °C (35 ng of Lys-C for each milligram of total protein lysate used in the first purification step). The urea concentration was then reduced to ≤2 m with Digestion Buffer (100 mm Tris, pH 8), and samples were incubated for 12–16 h with trypsin at 37 °C (30 ng of trypsin for each milligram of total protein lysate used in the first purification step). The supernatant was collected after gentle centrifugation of the bead solution (100 × g), and formic acid was added to a final concentration of 1% to end the digestion. Peptides were extracted further from the streptavidin beads with half the bead volume of 25% acetonitrile, 0.1% formic acid. The supernatant and extracts were pooled, concentrated using a SpeedVac, and acidified by 0.1% formic acid prior to mass spectrometric analysis. For one-dimensional (1-D) LC MS/MS analysis, the tryptic digest was directly injected onto the column, whereas the Lys-C/trypsin digests were desalted first with a C18 Ziptip (Millipore) before analysis. 1-D LC MS/MS was carried out by nanoflow reverse phase LC (Ultimate, LC Packings-Dionex) coupled on line to a quadrupole-orthogonal-time-of-flight tandem mass spectrometer (QSTAR XL, Applied Biosystems/MDS Sciex). Reverse phase LC was performed using a PepMap column (75-μm inner diameter × 150 mm long, LC Packings-Dionex), and the peptides were eluted using a linear gradient of 0–35% solvent B in 100 min at a flow of 200 nl/min. Solvent A contained 98% H2O, 2%acetonitrile, 0.1% formic acid; solvent B was composed of 98% acetonitrile, 2% H2O, 0.1% formic acid. The QSTAR mass spectrometer was operated in an information-dependent mode in which each full MS scan was followed by three MS/MS scans where the three most abundant peptide molecular ions were dynamically selected for CID, thus generating tandem mass spectra. In general, the ions selected for CID were the most abundant in the MS spectrum except that singly charged ions were excluded, and dynamic exclusion was used to prevent repetitive selection of the same ions within a preset time. Collision energies were programmed to be adjusted automatically according to the charge state and mass value of the precursor ions. To increase the number of the MS/MS spectra acquired from any given sample and improve the dynamic range of mass spectrometric analysis, multiple LC MS/MS runs were performed on the same sample with exclusion lists generated from the previous LC MS/MS runs using the Mascot script within the Analyst program. For the two-dimensional LC MS/MS, the digests were first separated by strong cation exchange (SCX) chromatography, which was performed using an AKTA system (Amersham Biosciences). Solvent A (5 mm KH2PO4, 30% acetonitrile, pH 3 adjusted with formic acid) and solvent B (solvent A with 350 mm KCl) were used to develop a salt gradient. The digests were separated by SCX chromatography by using a 2.1 × 10-mm polysulfoethyl A Guard column (Poly LC, Columbia, MD) at a flow rate of 150 μl/min, and the separation profile was monitored by UV absorbance at 215, 254, and 280 nm, respectively. A typical separation used 0% B from 0 to 10 min to allow for sample loading and removal of non-peptide species followed by a gradient of 0–100% B from 25 to 30 min. Fractions were manually collected based on UV absorbance. All the SCX fractions were desalted offline using a C18 Ziptip (Millipore) prior to LC MS/MS. The monoisotopic masses (m/z) of both parent ions and their corresponding fragment ions, parent ion charge states (z), and ion intensities from the MS/MS spectra acquired were automatically extracted using the script in the Analyst software and directly submitted for automated database searching for protein identification using two different search engines, Protein Prospector (University of California San Francisco) and Mascot (Matrix Science), to improve confidence levels of the protein identifications in the large datasets. The LC-Batchtag program within the developmental version of Protein Prospector was used for database searching. The mass accuracy for parent ions and fragment ions were set as ±100 and 300 ppm, respectively. An in-house Mascot program was also used for the database searching, and the mass accuracy for parent ions were set as ±100 ppm, and 0.3 Da was the fragment ion mass tolerance. The Swiss-Prot public database was queried to identify the purified proteins. In addition, the Search Compare program within the developmental version of Protein Prospector (14Chalkley R.J. Baker P.R. Huang L. Hansen K.C. Allen N.P. Rexach M. Burlingame A.L. Comprehensive analysis of a multidimensional liquid chromatography mass spectrometry dataset acquired on a quadrupole selecting, quadrupole collision cell, time-of-flight mass spectrometer: II. New developments in Protein Prospector allow for reliable and comprehensive automatic analysis of large datasets.Mol. Cell Proteomics. 2005; 4: 1194-1204Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) was used to make a list of proteins that differed between samples. Only the peptides identified with peptide score ≥25 in both Protein Prospector and Mascot search results were included for protein identification. Selected proteins were further validated by manual inspection of the MS/MS spectra. All the MS/MS spectra for the identified ubiquitinated peptides shown in Fig. 6 were manually examined. To search the existing databases of protein annotations, Gene Ontology, and protein interactions, a Web interface (contact14.ics.uci.edu/swisstogene.html) was developed to convert the Swiss-Prot accession numbers into gene names and yeast ORFs using the ExPASy (Expert Protein Analysis System) proteomics server of the Swiss Institute of Bioinformatics (SIB). This interface is built with Perl Common Gateway Interface (CGI) scripts. Furthermore a suite of Perl programs was used to process the output of our experiments, query the GOnet database (contact5.ics.uci.edu/gonet/sgdindex.html) (15Irwin B. Aye M. Baldi P. Beliakova-Bethell N. Cheng H. Dou Y. Liou W. Sandmeyer S. Retroviruses and yeast retrotransposons use overlapping sets of host genes.Genome Res. 2005; 15: 641-654Crossref PubMed Scopus (71) Google Scholar), and format the output from GOnet. GOnet is a database of yeast protein interactions and Gene Ontology that allows users to rapidly cluster genes according to interaction and GO hierarchy. With the information retrieved from GOnet, we were able to rapidly classify the proteins according to their molecular functions, biological processes, and cellular components. There are only two affinity purification strategies we are aware of that tolerate fully denaturing conditions such as 8 m urea or 6 m guanidinium chloride, namely binding of a hexahistidine sequence to Ni2+-chelating resins and the interaction between biotin and the Streptomyces avidinii protein streptavidin. We therefore thought to combine these two purification steps into a tandem affinity tag to develop a two-step purification strategy that is compatible with completely denaturing conditions. To this end we combined a RGSH6 tag with a bacterially derived polypeptide that serves as a biotinylation signal in vivo (16Cronan Jr., J.E. Biotination of proteins in vivo. A post-translational modification to label, purify, and study proteins.J. Biol. Chem. 1990; 265: 10327-10333Abstract Full Text PDF PubMed Google Scholar) (Fig. 1). We refer to this tandem affinity tag as the HB tag. The nine-amino acid-long RGSH6 peptide was chosen because it combines affinity to Ni2+ chelate resins and immunodetection with commercially available antibodies with low cross-reactivity toward proteins present in eukaryotic cells. A specific lysine residue in the biotinylation signal serves as an efficient acceptor for biotin in vivo. Biotinylation is catalyzed by endogenous biotin ligases, which are present in all prokaryotic and eukaryotic cells. We tested several HB-tagged proteins in the yeast Saccharomyces cerevisiae as well as in human cell lines and found that the HB tag was quantitatively biotinylated in vivo (see below). In addition, no effect on cell morphology or cell viability was observed when eight different essential yeast genes were replaced by HB-tagged alleles indicating that the HB tag generally does not interfere with protein function (data not shown). HB-tagged proteins can be sequentially purified by Ni2+ chelate chromatography and binding to streptavidin resins. The extraordinarily high affinity between biotin and streptavidin (Kd = 10−15 m) tolerates extremely stringent wash conditions such as 4% SDS, 8 m urea, organic solvents (e.g. 20% isopropanol/ethanol), and high salt conditions and thus guarantees highly purified samples (Ref. 17Savage D. Mattson G. Desai S. Niedlander G. Morgensen S. Conklin E. Avidin-Biotin Chemistry: A Handbook. 2nd Ed. Pierce Chemical, Rockford, IL1994Google Scholar and data not shown). The high affinity between biotin and streptavidin hampers efficient elution of HB-tagged proteins from streptavidin beads. This does not present a problem for mass spectrometric analyses because Lys-C and trypsin digestion is efficient on bead-bound proteins (data not shown and Refs. 18Kho Y. Kim S.C. Jiang C. Barma D. Kwon S.W. Cheng J. Jaunbergs J. Weinbaum C. Tamanoi F. Falck J. Zhao Y. A tagging-via-substrate technology for detection and proteomics of farnesylated proteins.Proc. Natl. Acad. Sci. U S A. 2004; 101: 12479-12484Crossref PubMed Scopus (280) Google Scholar and 19Rybak J.N. Ettorre A. Kaissling B. Giavazzi R. Neri D. Elia G. In vivo protein biotinylation for identification of organ-specific antigens accessible from the vasculature.Nat. Methods. 2005; : 291-298Crossref PubMed Google Scholar). Importantly streptavidin itself proved to be largely resistant to Lys-C and trypsin digestion under these conditions. Typically less than 1% of the detected peptides resulted from fragmentation of streptavidin, which did not interfere with mass spectrometric analyses. For applications where elution of purified proteins is required, we inserted a TEV protease cleavage site that allows proteolytic release of the bound protein from streptavidin beads (Fig. 1A). In addition, we constructed additional derivatives of the HB tag that are useful for expression of amino-terminal or carboxyl-terminal fusion proteins (Fig. 1A). A second hexahistidine sequence was included to form the HBH tag, which increases efficiency of the first purification step by Ni2+ chelate chromatography and is particularly useful for purification of low abundant proteins. We first tested expression and purification of the yeast transcriptional activator Met4 fused at its carboxyl terminus to the HBH tag (Met4HBH). Met4 is posttranslationally modified by a ubiquitin chain, which is easily lost during native purification and is therefore well suited to test preservation of modifications during HB-based fractionation (20Flick K. Ouni I. Wohlschlegel J.A. Capati C. McDonald W.H. Yates J.R. Kaiser P. Proteolysis-independent regulation of the transcription factor Met4 by a single Lys 48-linked ubiquitin chain.Nat. Cell Biol. 2004; 6: 634-641Crossref PubMed Scopus (127) Google Scholar21Kaiser P. Flick K. Wittenberg C. Reed S.I. Regulation of transcription by ubiquitination without proteolysis: Cdc34/SCF(Met30)-mediated inactivation of the transcription factor Met4.Cell. 2000; 102: 303-314Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar22Kuras L. Rouillon A. Lee T. Barbey R. Tyers M. Thomas D. Dual regulation of the met4 transcription factor by ubiquitin-dependent degradation and inhibition of promoter recruitment.Mol. Cell. 2002; 10: 69-80Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Met4HBH was expressed under control of its own promoter and, like most transcription factors, is of relatively low abundance. Cells were lysed in a buffer containing 8 m urea and incubated with a Ni2+ chelate resin (Fig. 2A). Detection of Met4HBH with antibodies directed against the RGSH6 epitope, which is part of the HBH tag, showed efficient binding to the resin (Fig. 2A, left panel). Met4HBH was subsequently eluted with a buffer containing 8 m urea and a combination of components that block the interaction of the hexahistidine sequence with the Ni2+ chelate resin (2% SDS, 10 mm EDTA, pH = 4.3). The pH of the eluate was readjusted to 7.5 and incubated with streptavidin-agarose. Close to 100% of Met4HBH was retained on the streptavidin resin (Fig. 2A). Eukaryotic cells have between four and six endogenous biotinylated proteins (23Chapman-Smith A. Cronan Jr., J.E. The enzymatic biotinylation of proteins: a post-translational modification of exceptional specificity.Trends Biochem. Sci. 1999; 24: 359-363Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). The first purification step by Ni2+ chelate chromatography efficiently removed these. This was evident by comparing load (L) and flow-through (FT) lanes in the Fig. 2A, right panel, where all biotinylated proteins were detected using a streptavidin-horseradish peroxidase (HRP) conjugate. Met4HBH and its modified forms were the only biotinylated proteins that were retained on the Ni2+ chelate resin and thus specifically separated from endogenous biotinylated proteins (Fig. 2, right panel). Importantly, immunoblot analysis of the purification steps with an antibody directed against the RGSH6 epitope (Fig. 2A, left panel) demonstrated greater than 90% in vivo biotinylation efficiency of the HBH tag because most of the Met4HBH was retained on the streptavidin resin indicating the presence of biotin on Met4HBH (Fig. 2A). Similar results were obtained with several other yeast proteins (Rpt1, Rpt5, Rpn11, Pre1, Pre10, Skp1, and Smt3), including proteins that were overexpressed from the GAL1 promoter (Met30, Cdc4, Met32, and Met31) and accumulated to significantly higher levels than Met4 (data not shown). In all cases in vivo biotinylation appeared to be close to 100%, demonstrating effective recruitment of endogenous biotin ligases by the HB tag. Silver staining of the different purification fractions demonstrated the dramatic reduction of the background as compared with single step purification by Ni2+ chelate chromatography (Fig. 2B). Accordingly mass spectrometric analysis showed that nonspecific purification was reduced more than 6-fold (to 16%) by the second purification step on streptavidin beads as compared with the single step purification on Ni2+ chelate resin (data not shown). This represents a significant improvement of protein purification under de
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