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Plasmid 59 (2008) 54–62

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Tandem affinity purification vectors for use in grampositive bacteria

Xiao Yang, Geoff P. Doherty, Peter J. Lewis *

School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW 2308, Australia

Received 20 July 2007, revised 17 October 2007

Communicated by Julian Rood

Abstract

Tandem affinity purification has become a valuable tool for the isolation of protein complexes. Here we describe theconstruction and use of a series of plasmid vectors for Gram positive bacteria. The vectors utilize the SPA tag as wellas variants containing a 3C rather than the TEV protease site as 3C protease has been shown to work efficiently at thelow temperatures (4 �C) used to isolate protein complexes. In addition, a further vector incorporates a GST moiety in placeof the 3 · FLAG of the SPA tag which provides an additional tagging option for situations where SPA binding may beinefficient. The vectors are all compatible with previously constructed fluorescent protein fusion vectors enabling construc-tion of a suite of affinity and fluorescently tagged genes using a single PCR product.Crown copyright � 2007 Published by Elsevier Inc. All rights reserved.

Keywords: Tandem affinity purification; Plasmid vectors; Bacteria; Bacillus subtilis; RNA polymerase

1. Introduction

The purification and characterization of proteincomplexes has become an increasingly importantfacet of proteomic analysis in the last few years.Many approaches have been developed for suchstudies including yeast 2-hybrid (Fields and Song,1989), bacterial 2-hybrid (Karimova et al., 1998),immunoprecipitation (e.g. Heinrich et al., 2006)and tandem affinity tag purification (TAP-tag;Rigaut et al., 1999; Puig et al., 2001). Although ini-tially it was hoped that one single approach wouldpermit the exhaustive analysis of all protein–protein

0147-619X/$ - see front matter Crown copyright � 2007 Published by

doi:10.1016/j.plasmid.2007.11.001

* Corresponding author. Fax: +61 2 4921 6923.E-mail address: Peter.Lewis@newcastle.edu.au (P.J. Lewis).

interactions, it is now clear that several approachesare required to ensure the maximum coverage. Nev-ertheless, approaches such as TAP-tag have provedvery useful for the identification of relatively stableprotein complexes, and have helped in the identifi-cation of novel interactions (e.g. Forler et al.,2003; Knuesel et al., 2003; Zeghouf et al., 2004;Gould et al., 2004; Rohila et al., 2004; Butlandet al., 2005; Rubio et al., 2005). In addition to pro-teomic analysis, considerable information has beenobtained from the analysis of protein dynamicswithin cells using fluorescent protein tags (Lewis,2004; Giepmans et al., 2006).

In this paper, we describe the construction andtesting of a series of vectors for the tandem affinitypurification of protein complexes from the model

Elsevier Inc. All rights reserved.

X. Yang et al. / Plasmid 59 (2008) 54–62 55

Gram positive bacterium Bacillus subtilis. In addi-tion to utilizing the well characterized SPA-tag(Zeghouf et al., 2004; Table 1), we also constructeda novel tag comprising the glutathione-S-transferasegene (GST) preceeded by a protease cleavage siteand a calmodulin binding protein (CBP) moiety,and have called this gTAP. These vectors permitthe production of four different tagged variants ofa protein, enabling a choice of protease cleavage siteand epitopes used for the affinity purification of pro-tein complexes. The vectors are also similar to onespreviously described for the construction of fluores-cently tagged proteins (Lewis and Marston, 1999;Feucht and Lewis, 2001), and so the product of asingle PCR reaction can be used to produce multiplefluorescent or tandem affinity purification tags.

Table 1Plasmids and strains use in this study

Plasmids/strains

Genotype

pJL148 bla CBP TEV 3 · FLAG

pCm:Sp bla (cat0-spc-’cat)pST3 bla cat Pxyl

0rpoC gfp

pNG76 bla cat Pxyl greA gfp

pSG1164 bla cat Pxyl gfp

pGEX-6P2 bla Pspac lacI GST

pNG207 bla cat Pxyl CBP TEV 3 · FLAG

pNG208 bla cat Pxyl CBP 3C 3 · FLAG

pNG214 bla cat Pxyl CBP TEV GST

pNG218 bla cat Pxyl CBP 3C GST

pNG210 bla cat Pxyl0rpoC CBP 3C 3 · FLAG

pNG217 bla cat Pxyl greA CBP 3C 3 · FLAG

pNG221 bla cat Pxyl0rpoC CBP 3C GST

pNG498 bla cat Pxyl0nusB CBP 3C 3 · FLAG

pNG560 bla cat Pxyl0nusB-CBP 3C GST

E. coli DH5a F� endA1 hsdR17 supE44 thi-1 k� recA1 gyrA96 relA1

argF)U169 /80 dlacZ DM15

B. subtilis

168trpC2

BS61 trpC2 chr::pNG76 (nusB-gfp cat)BS425 trpC2 chr::pNG76 (nusB-gfp cat)XpCm:SpBS400 trpC2 chr::pNG210 (rpoC-CBP 3C 3 · FLAG cat)BS401 trpC2 chr::pNG217 (greA-CBP 3C 3 · FLAG cat)BS428 trpC2 chr::pNG221 (rpoC-CBP 3C GST cat)BS434 trpC2 chr::pNG217 (greA-CBP 3C 3 · FLAG cat) chr:

(nusB-gfp cat)XpCm:SpBS438 trpC2 chr::pNG498 (nusB-CBP 3C 3 · FLAG cat)BS468 trpC2 chr::pNG560 (nusB-CBP 3C GST cat)BS469 trpC2 chr::pNG221 (rpoC-CBP 3C GST cat) chr::pNG

cat)XpCm:Sp

bla, cat, sp; ampicillin, chloramphenicol, spectinomycin resistance generespectively.

The vectors have been designed so that constructsintegrate into the chromosome by single crossoverensuring the level of expression of the tagged fusionis similar to the wild-type protein. Furthermore, thevectors should be compatible unmodified, or withminimal modification, for use in a wide variety oftransformable Gram positive bacteria.

2. Materials and methods

2.1. Bacterial strains and media

A list of bacterial strains and plasmids is presented inTable 1. All cloning was carried out in Escherichia coli

DH5a (Gibco-BRL). Strains were grown on nutrient agar(NA) supplemented with the appropriate antibiotics

Source/construction

Zeghouf et al. (2004)Bacillus Genetic Stock CenterLewis et al. (2000)Doherty et al. (2006)Lewis and Marston (1999)GE HealthcareSPA-tag cloned into pSG11643C linker cloned into pNG207GST cloned into pNG207GST cloned into pNG208XhoI–EcoRI fragment of 0rpoC from pST3 intosimilar cut pNG208XhoI–EcoRI fragment of greA from pNG76 intosimilar cut pNG208XhoI–EcoRI fragment of 0rpoC from pST3 intosimilar cut pNG2180nusB cloned into Acc65I and XhoI cut pNG2080nusB cloned into Acc65I and XhoI cut pNG218

D (lacZYA- Gibco-BRL

Laboratory Stock

Doherty et al. (2006)BS61 transformed with pCm:SppNG210 transformed into 168pNG217 transformed into 168pNG221 transformed into 168

:pNG425 BS425 chromosomal DNA transformed intoBS401pNG498 transformed into 168pNG560 transformed into 168

76 (nusB-gfp BS425 transformed with pNG221

s, respectively. Pxyl, Pspac, xylose and IPTG inducible promoters,

56 X. Yang et al. / Plasmid 59 (2008) 54–62

(ampicillin, 100 lg/ml; chloramphenicol, 5 lg/ml; specti-nomycin, 50 lg/ml), and cultured in LB medium (Sam-brook et al., 1989). Where used, xylose was added to0.5% (w/v) in both solid and liquid media. All bacterialstrains were grown at 37 �C unless otherwise stated.B. subtilis transformations were performed as describedby Anagnostopoulos and Spizizen (1961) as modified byJenkinson and Mandelstam (1983) or as described byAlbano et al. (1987).

2.2. Plasmid construction

The SPA-tag sequence was amplified from pJL148(Zeghouf et al., 2004) using the following primers 5 0-TCGCCTGCAGATGGAAAAGAG-3 0 and 5 0-GAT-ATCTCCCCACTAGTCACTACTTG-3 0. The resultingproduct was cut with PstI and SpeI, and ligated into sim-ilarly cut pSG1164 (Lewis and Marston, 1999) to replacethe gfp gene with the SPA-tag to give pNG207 (Table 1;Fig. 1).

pNG207 was digested with NheI and SacI to remove theTEV site and the 3C protease site introduced using the link-ers 5 0-CTAGCCTGGAAGTGCTGTTTCAGGGCCCGGAGCT-3 0 and 5 0-CCGGGCCCTGAAACAGCACTTCCAGG-3 0 that had been annealed and phosphorylatedusing T4 polynucleotide kinase (Sambrook et al., 1989) togive pNG208 (Table 1; Fig. 1).

To produce the gTAP vector, the GST sequence wasamplified from pGEX-6P2 (GE Healthcare) using theprimers 5 0-CAGGAAACAGAGCTCATGTCCCCT-3 0

and 5 0-GGAACAGAACTAGTTAATCCGATT-3 0. Theresulting product was cut with SacI and SpeI and insertedinto similarly cut pNG207 and pNG208, replacing the3 · FLAG moiety, to give pNG214 and pNG218, respec-tively (Table 1; Fig. 1).

A 600 bp fragment corresponding to the 3 0 terminalregion of rpoC was subcloned from pST3 (Lewis et al.,2000) into XhoI–EcoRI cut pNG208 and pNG218 to pro-duce pNG210 and pNG221, respectively (Table 1). Theentire greA gene was removed from pNG76 (Dohertyet al., 2006) using XhoI and EcoRI and inserted into simi-larly cut pNG208 to give pNG217. A 371 bp fragment cor-responding to the 3 0 region of nusB was PCR amplifiedusing primers 5 0-GAAAAAGCTGGTACCGCACTATTT-3 0 and 5 0-GGGTTCCTCGAGTGATTGTTCAAT-3 0

and cloned into Acc65I and XhoI cut pNG208 to givepNG498.

All constructs were checked by restriction digest andsequencing.

2.3. Tandem affinity purification and protein detection

Tagged B. subtilis strains were grown up in 1 L LB toan A600 1.0–1.4 prior to harvesting and cell pellets werestored at �80 �C. Two different lysis procedures were usedin order to break open cells. Firstly, cells were lysed by

resuspension in 10 ml TES (20 mM Tris–HCl, pH 7.0,100 mM NaCl, 5 mM EDTA) supplemented with0.5 mg/ml lysozyme (Sigma–Aldrich) followed by incuba-tion at 37 �C for 5 min. The highly viscous lysate was thenplaced on ice and briefly sonicated to fragment the DNAand reduce viscosity. The lysate was then clarified by cen-trifugation at 8000g at 4 �C for 25 min. The secondinvolved resuspending cells in 10 ml of TES and passingthis three times through a French press at 25,000 psibefore clarifying the lysate by centrifuging at 8000g for10 min at 4 �C.

One hundred microliters of anti-FLAG M2 resin(Sigma–Aldrich) pre-equilibrated in TBS (50 mM Tris–HCl, pH 7.5, 150 mM NaCl) was added to the clarifiedlysate and the mixture rotated at 4 �C for 3 h. The resinwas poured into a Micro Bio-Spin column (BioRad) andthen washed with 20 column volumes TBS. The settledresin was then resuspended in 500 ll 3C cleavage buffer(50 mM Tris–HCl, pH 7.0, 150 mM NaCl, 0.1% Triton-X 100) and the column capped following the addition of3C protease (7 ll, 6.5 mg/ml) and rotated at 4 �C for4 h. The sample was eluted from the column into a1.5 ml tube containing 500 ll CBP buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 2 mM CaCl2). Additional0.2 M CaCl2 was added to bring the final Ca2+ concentra-tion to 2 mM. 200 ll of pre-equilibrated calmodulin-sepharose 4B resin (GE Healthcare) was added and themixture rotated overnight at 4 �C. The equilibrated mix-ture was poured into a fresh column, and the resin washedwith 10 column volumes of CBP buffer prior to elutionwith 4 column volumes elution buffer (50 mM Tris–HCl,pH 7.5, 150 mM NaCl, 2 mM EGTA). Following SDS–PAGE Western blots were performed using rabbit poly-clonal anti-RpoC (b 0 subunit of RNA polymerase),NusA, GreA, rA, GFP and FtsZ antibodies.

For the tandem affinity purification using gTAP-tagged complexes, 200 ll of glutathione agarose beads(Sigma–Aldrich) pre-equilibrated in PBS (10 mMNaH2PO4, 150 mM NaCl, pH 7.4) was added to the clar-ified lysate and rotated at 4 �C for 3 h. The resin waspoured into a Micro Bio-Spin column (BioRad) and thenwashed with 20 column volumes PBS. All subsequentpurification steps were performed as detailed above.

3. Results and discussion

3.1. Production of tagging vectors

Details of the four vectors produced for affinitytagging which provide flexibility in choosing the opti-mal system for a particular purification protocol areshown in Fig. 1A. All of the vectors are based onthe gfp tagging vector pSG1164 (Lewis and Marston,1999), and so digestion of a single PCR product canbe used to produce multiple tagged variants of a gene.

Fig. 1. Tandem affinity purification vectors. The plasmid map and protease cleavage site junctions are shown in (A). The map of the gTAPvectors pNG214/218 with the sequence of the multiple cloning site for all vectors is shown at the top of the panel. The DNA and singleletter protein sequences corresponding to the region encompassing the protease cleavage sites for all of the vectors is shown at the bottomof the panel. (B) A schematic for how the vectors integrate into the chromosome by single crossover creating a full-length fusion under thecontrol of the wild-type promoter, and control of expression of any genes within an operon that may lay downstream of the site ofinsertion. Pwt, wild-type promoter; Pxyl, xylose inducible promoter; ori, origin of plasmid replication (only active in E. coli); bla, geneencoding b-lactamase; cat, gene encoding chloramphenicol acetyl transferase.

X. Yang et al. / Plasmid 59 (2008) 54–62 57

pNG207 and 208 were designed for SPA tagging witheither a TEV (pNG207) or 3C (pNG208) proteasecleavage site. Likewise, pNG214 and 218 permitgTAP tagging with either TEV (pNG214) or 3C(pNG218) protease sites. Assembly of the tags ismodular so that if required, epitopes can also be read-ily changed: The CBP moiety is inserted betweenunique PstI and NheI sites, whereas the 3 · FLAG

or GST moiety resides between unique SacI and SpeIsites.

These vectors have been principally designed foruse in B. subtilis but will be applicable to any readilytransformable Gram positive bacterium making thevectors useful tools for proteomics-based projects.Briefly, since the vectors are unable to replicate inGram positive organisms, providing a suitable

Fig. 2. The 3C protease efficiently cleaves a GreA–SPA taggedfusion at low temperatures. Samples were immunoblotted withanti-GreA antibodies. Lane 1, supernatant of cleared lysate ofstrain BS401 (S). Lane 2, flow through following binding ofsupernatant to M2 beads (M2 FT). Lane 3, wash from M2 beads(M2 wash). Lane 4, elution from M2 beads following cleavagewith 3C protease (3C EL). Lane 5, acid glycine wash to determinethe level of uncleaved and un-eluted cleaved GreA–SPA taggedfusion remaining on M2 beads (M2 Acid Wash).

58 X. Yang et al. / Plasmid 59 (2008) 54–62

region of homology has been inserted (around500 bp), the vectors will recombine by single cross-over (Campbell integration) with the chromosomeof the host resulting in a partial duplication of thetarget gene (Fig. 1B). The full-length copy of thetarget gene, transcribed from its native promoter,will carry the affinity tag at the 3 0 end producing aC-terminal fusion, and the partially duplicated 3 0

fragment will be preceded by the Pxyl promoter.This permits xylose-inducible control of expressionof any genes that lie downstream of the target genewithin the same operon in organisms containing axylR gene (Lewis and Marston, 1999; Dohertyet al., 2006). In organisms that do not contain axylose repression system, so long as Pxyl is recog-nized, downstream genes will be constitutivelyexpressed. In vitro transcription assays using RNApolymerase purified from both B. subtilis andE. coli resulted in transcripts of the expected lengthshowing that the Pxyl promoter can be recognizedby RNA polymerase from distantly related bacteria(data not shown).

3.2. Establishment of 3C protease in SPA tag

cleavage

To date, the majority of tandem affinity tag sys-tems have made use of the TEV protease (e.g. Puiget al., 2001; Rohila et al., 2004). Although consider-able success has been achieved using these systems,we wanted to design vectors that maximized flexibil-ity of use by providing an alternative protease cleav-age site. Therefore, we decided to determine if the3C protease that is known to work efficiently atlow temperatures (Rubio et al., 2005) representeda suitable alternative in affinity purification systems.

Strain BS401 containing a fusion to the transcrip-tion elongation factor GreA (greA-SPA; Table 1)was grown and lysed as detailed in Materials andMethods and the results of SPA tag binding toanti-FLAG M2 beads and efficiency of 3C cleavageare shown in Fig. 2. Due to the small size of GreA(17 kDa) molecular weight changes on removal ofthe 3 · FLAG tag (approx. 3 kDa) to determinecleavage efficiency could be readily determined.Thus, the GreA–SPA fusion had a molecular weightof around 25 kDa, whereas the cleaved GreA–CBPproduct had a molecular weight of around22 kDa. Comparison of the samples in the GreAimmunoblot shows that the eluate from the M2beads following 3C cleavage generated a smallerfragment than prior to cleavage (compare lanes 1–

3 with lane 4; Fig. 2). Washing the beads with0.2 M glycine pH 2.2 to remove any remaining pro-tein indicated that little protein remained bound tothe beads and that the cleavage reaction had beenhighly efficient (compare lanes 4 and 5; Fig. 2).We estimate that under the conditions used in thisexperiment, cleavage was at least 90% efficientshowing that 3C protease is an effective alternativeto TEV in tandem affinity purification.

3.3. Comparison of SPA and gTAP tags

Due to the location of tags within biologically rel-evant complexes, there will be times when a small tagis prevented from interacting efficiently with a resindue to steric hindrance and so a larger tag shouldprovide better binding and recovery of the complex.Likewise, there will also be situations where a largetag may inhibit the efficient binding of some proteinswithin a complex or render the fusion non-func-tional so a smaller tag will be required. It is also pos-sible that due to empirical, currently not understoodprocesses, one tag may simply be more efficient atrecovering a complex than another. Therefore, wewished to provide flexibility to our system enablingthe rapid construction and testing of multiple tagsin order that the most efficient recovery of complexescould be rapidly determined.

In previous studies we have found that the choiceof resin is extremely important when purifyingRNA polymerase (RNAP) containing a 6 or 9-histi-dine tag at the C-terminus of the b 0 subunit (notshown). The crystal structure of RNAP shows thatthe C-terminus of the b 0 subunit is solvent exposedand should theoretically be available to bind toaffinity resins when fused to a suitable tag (Zhanget al., 1999; MacDougall et al., 2005). Indeed,

X. Yang et al. / Plasmid 59 (2008) 54–62 59

GFP fusions to the C-terminus of this subunit inboth B. subtilis and E. coli have been shown to befully functional (Lewis et al., 2000; Cabrera andJin, 2003a,b). Currently, there is only a single sourceof beads for binding the FLAG tag (the first tagused in the SPA tag system), and so we constructedstrains containing fusions of the RNAP b 0 subunitto either the SPA tag or gTAP tag (strains BS400and BS428, respectively, Table 1). Strains weregrown and processed identically (apart from the ini-tial binding to and elution from either M2 or gluta-thione beads), and the final purified complexesimmunoblotted for the presence of RpoC (the b 0

subunit of RNAP) and transcription elongation fac-tor GreA (Fig. 3). The fractions from strain BS400(SPA-tag) gave undetectable signals for either RpoCor GreA (Fig. 3, lanes 5–8) indicating that very littleor no complex had been purified. Subsequent anal-ysis indicated that this was due to the extremely lowefficiency of binding of the complex to the M2 beads(not shown). Conversely, the fractions from strainBS428 (gTAP-tag) gave strong RpoC signals, andsmall, but readily detectable GreA signals indicatingthis fusion can be used for the isolation of transcrip-tion complexes (Fig. 3, lanes 1–4). Thus, the choiceof tag can lead to profound differences in the recov-ery of protein complexes.

3.4. Purification of protein complexes

Strain BS434 (greA-SPA; nusB-gfp; Table 1) wasused to test the value of the vectors in the purifica-tion of transcription complexes. In previous workwe have shown that the subunit composition of m-and rRNA elongation complexes is different withmRNA complexes containing NusA, NusG andGreA, whereas rRNA complexes contain NusA,

Fig. 3. Comparison of the recovery of gTAP and SPA-tagged RNA pWestern blots were immunostained to detect the b 0 subunit of RNA po(kDa) are shown down the left hand side of the blot. Lanes 1–4, sepolymerase; lanes 5–8, similar elutions of SPA-tagged RNA polymeras

NusG, NusB and NusE (Davies et al., 2005; Doher-ty et al., 2006). Strain BS434 could, therefore, beused to help confirm the composition of these elon-gation complexes as complexes containing GreAshould have no, or very little, NusB.

Following SPA-tag purification, samples wereimmunoblotted using polyclonal anti-RpoC (b 0 sub-unit of RNAP), NusA, GreA, rA, GFP and FtsZantibodies (Fig. 4A). Since GreA is a transcriptionelongation factor, and rA is an initiation factor wewould expect that complexes purified from BS434should not contain any initiation factors. A strongsignal for GreA was obtained (lane 1, Fig. 4A), aswould be expected since this protein was SPA-tagged, indicating efficient binding and recovery.Signals for RNAP and elongation factor NusA werealso obtained (lanes 2 and 3, Fig. 4A) showing thatSPA-tagged GreA can be used to isolate transcrip-tion elongation complexes. No signal could bedetected for the NusB–GFP fusion (lane 5,Fig. 4A) suggesting that through tagging GreA itmay be possible to specifically isolate transcriptioncomplexes involved in mRNA synthesis, consistentwith the cytological data of Doherty et al. (2006)that indicated NusB is restricted to sites of rRNAsynthesis. The abundant cell division protein FtsZ,used as a negative control, also gave no detectablesignal (lane 6, Fig. 4A) indicating that transcriptioncomplexes were being specifically isolated.

Additional experiments were performed to con-firm this hypothesis using strain BS469 (RpoC–gTAP, NusB–GFP; Fig. 4B), BS438 (NusB–SPA)and BS468 (NusB–gTAP). When using strainBS469 NusA and GreA could all be detected, butnot the NusB–GFP fusion (lanes 2–4, Fig. 4B). Signalwas also absent in the FtsZ negative control (lane 5,Fig. 4B). In addition, when performing isolation of

olymerase in complex with transcription elongation factor GreA.lymerase (Anti-RpoC) and GreA (Anti-GreA). Molecular weightsrial elutions from calmodulin-sepharose of gTAP-tagged RNAe.

Fig. 4. Isolation of transcription elongation complexes. (A) Western blots of complexes isolated from strain BS401 (GreA–SPA) wereimmunostained to detect GreA (lane 1), RNA polymerase (RNAP, b 0 subunit of RNA polymerase, lane 2), NusA (lane 3), rA (lane 4),NusB–GFP (lane 5) and FtsZ (lane 6). (B) Western blots of complexes isolated from strain BS469 (RpoC–gTAP, NusB–GFP). RNAP(lane 1), GreA (lane 2), NusA (lane 3), NusB–GFP (lane 4) and FtsZ (lane 5). Molecular weights (kDa) are shown on the left hand side.The image represents a composite of separately immunostained strips, aligned through reference to molecular weight markers run in lanesadjacent to each sample.

60 X. Yang et al. / Plasmid 59 (2008) 54–62

complexes with the NusB–SPA or gTAP strainsBS438 and BS468, respectively, no RNAP or NusAcould be detected (not shown). NusB does notdirectly interact with RNAP. Rather, it forms a het-erodimer with NusE (ribosomal protein S10) andbinds to RNAP via NusE in co-complex with theboxA region of an rRNA transcript (Greive et al.,2005). Thus, it appears that a limitation of theTAP-tag system is in the isolation of nucleoproteincomplexes containing RNA. Multiple attempts weremade to isolate NusB-containing complexes, but inthe absence of fixation it could not be reliablydetected. Studies in E. coli have also failed to isolateNusB in transcription complexes (Butland et al.,2005). It may be possible to refine protocols in thefuture so that nucleoprotein complexes can be iso-lated, but this may involve the addition of fixativesthat can also cause false positive results and so greatcare will be required to ensure artifacts areeliminated.

Surprisingly, when performing GreA–SPA purifi-cation, a weak signal was also obtained for rA (lane4, Fig. 4A) which was unexpected. Control experi-ments where extracts from untagged strains werepassed over the beads gave no detectable signalssuggesting that the presence of rA in the sampleswas specific rather than due to residual backgroundor non-specific binding events. It is possible thatsome rA is present in elongation complexes as ithas been reported in E. coli that r70 can remainassociated with RNAP during this stage of tran-scription (Bar-Nahum and Nudler, 2001; Mukho-padhyay et al., 2004). There are also reports thatGre proteins could play a role in transcription initi-ation (Hsu et al., 1995; Potrykus et al., 2006; Susaet al., 2006; Stepanova et al., 2007), and so the rA

detected could have arisen from initiation com-plexes, although other work has cast some doubton the role of Gre proteins in transcription initia-tion (Rutherford et al., 2007).

X. Yang et al. / Plasmid 59 (2008) 54–62 61

In this study, proteins were detected by immuno-blot and no attempt was made to perform massspectrometry on samples, although there is no rea-son to suggest these vectors would not be suitablefor such approaches for the identification of thecomposition of protein complexes.

In summary, we have designed and tested a seriesof modular tandem affinity purification vectors foruse in Gram positive bacteria that can be readilymodified, and are also compatible with a parallelseries of fluorescent protein fusion vectors enablingthe construction of multiple fusions from a singlePCR product.

Acknowledgments

The authors thank Liz Harry for providing anti-FtsZ antiserum. This work was funded by grantsprovided by the ARC (DP0664370), NHMRC (Pro-ject Grants 455597 and 455646) and DEST (ISLCG110055), and as part of the BaSysBio 6th Frame-work project.

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