A Method for the Rapid and Efficient Elution of Native
Affinity-Purified Protein A Tagged Complexes
Caterina Strambio-de-Castillia,†Jaclyn Tetenbaum-Novatt,†Brian S.Imai,‡Brian T.Chait,§and
Michael P.Rout*,†
The Rockefeller University,1230York Avenue,New York New York 10021-6399
Received May 24,2005
A problem faced in proteomics studies is the recovery of tagged protein complexes in their native and active form.Here we describe a peptide,Bio-Ox,that mimics the immunoglobulin G (IgG)binding interface of Staphylococcus aureus Protein A,and competitively displaces affinity-purified Protein A fusion proteins and protein complexes from IgG-Sepharose.We show that Bio-Ox elution is a robust method for the efficient and rapid recovery of native tagged proteins,and can be applied to a variety of structural genomi
cs and proteomics studies.
Keywords:Staphylococcus aureus •Protein A •affinity purification •proteomics •fusion protein
Introduction
Protein -protein interactions are central to the maintenance and control of cellular processes.The study of such protein -protein interactions has been greatly enhanced by fusion protein technology,wherein specific peptide or protein domain “tags”are fused to the protein of interest (generally at either its carboxyl-terminus or amino-terminus).These tags can facilitate the detection,increase the yield,and enhance the solubility of their associated proteins.1-3Most importantly,these fusion domains have been exploited to allow the single-step purification of the test protein either alone or in complexes with its in vivo binding partners.4-6The yield of these purifica-tion methods is often high enough to allow the identification of such binding partners by mass spectrometry.
A commonly used affinity tagging method generates ge-nomically expressed Protein A (PrA)fusion proteins by modify-ing the coding sequence of the protein under study via PCR-directed approaches.7-9This method takes advantage of the ∼10nM binding affinity of PrA from S taphylococcus aureus for the constant region (Fc)of immunoglobulin G (IgG).10After purification on IgG-conjugated resins,PrA-t
agged proteins or protein complexes are most commonly eluted from the resin using high or low pH conditions.These elution methods typically lead to the denaturation of the isolated proteins,the dissociation of complexes,and concomitant loss of activity.However,it is often desirable to recover soluble native protein or protein complexes.One method by which this can be achieved is by constructing a cleavable tag.Such tags carry a specific cleavage site for a protease placed proximal to the tagged protein,allowing the tag to be removed from the fusion protein.Proteases that are widely used for this purpose include
blood coagulation factors X (factor Xa),enteropeptidase (en-terokinase),alpha-thrombin,and the tobacco etch virus (TEV)protease.Nevertheless,this method has drawbacks.First,the literature is replete with reports of fusion proteins that were cleaved by these proteases at sites other than the canonical cleaving site.11-14Second,the removal of the tag destroys the ability to detect or further purify the protein of interest,necessitating the encumbrance of a second,tandem tag.15Here,we describe a rapid single step method for the efficient recovery of native and active PrA fusion proteins and protein complexes from IgG-Sepharose.This technique avoids the complications of having to use a protease and in addition has the advantage of retaining the original tag on the target protein after elution,permitting further purification steps and detection of the fusion protein in subsequent experimen
ts.Our method takes advantage of a previously described peptide,termed FcIII,16which mimics the protein -protein binding interface of PrA for the hinge region on the Fc domain of human IgG.We modified FcIII by the addition of a biotin moiety to its amino-terminus to increase the peptide’s solubility while leaving its affinity for Fc intact s making it a more effective elution reagent.We termed this modified peptide,Bio-Ox.
To investigate the properties of Bio-Ox,PrA-tagged proteins were isolated in their native state from yeast on an IgG-conjugated Sepharose resin,either alone or in combination with their in vivo interacting partners;the Bio-Ox peptide was then used to competitively displace the tagged proteins and elute them from the resin.The efficiency of elution was monitored by quantitatively comparing the amounts of proteins eluted to the amounts remaining on the resin under a variety of test conditions.We show that Bio-Ox elution is a robust method for the efficient and rapid recovery of native tagged proteins that can be applied to a variety of structural genomics and proteomics studies.
*To whom correspondence should be addressed.Tel:+1(212)327-8135.E-mail:kefeller.edu.†
Laboratory of Cellular and Structural Biology,Box 213.‡
Proteomics Resource Center,Box 105.§
Laboratory of Mass Spectrometry and Gaseous Ion Chemistry,Box 170.
2250Journal of Proteome Research 2005,4,2250-2256
10.1021/pr0501517CCC:$30.25
©2005American Chemical Society
Published on Web
10/08/2005
Experimental Section
Peptide Synthesis,Oxidation and Cyclization.Peptides were synthesized using standard Fmoc protocols.Typical deprotec-tion times with20%piperidine were2times10min and typical coupling times with4-10-fold excess of amino acids over resin were2to6h.Small batches of peptides were made on a Symphony synthesizer(Protein Technologies,Inc.),while larger batches were made manually.Peptides
were cleaved from the resin using94.5%trifluoroacetic acid, 2.5%water, 2.5% ethanedithiol and1%triisopropylsilane for3h at25°C.The solubilized peptides were precipitated with10volumes of cold tert-butyl methyl ether and the precipitated peptide was washed several times with ether prior to air-drying.The air-dried peptide was dissolved in20%acetonitrile in water to approximately0.5mg/mL,the pH was adjusted to8.5using sodium bicarbonate and the peptide was allowed to air oxidize overnight to promote cyclization.The progress of cyclization was monitored by mass spectrometry.The cyclized crude peptide was purified using standard preparative reversed phase HPLC using a Vydac218TP1022C18column.
Peptide Solubility.Eluting peptides were suspended at a concentration of440µM(0.77mg/mL for BioOx;0.67mg/mL for FcIII),in peptide buffer by extensive vortexing.The peptide concentration was verified by measuring the OD280nm of each solution(extinction coefficient:1OD280nm)0.13mg/mL).The peptide solutions/suspensions were then combined with equal amounts of a100mM buffer to obtain∼220µM peptide at a range of pH values(buffers:Na-Acetate pH4.8,Na-Citrate pH 5.4,Na-Succinate pH5.8,Na-MES pH6.2,BisTris-Cl pH6.5, Na-HEPES pH7.4,Na-TES pH7.5,Tris-Cl pH8.3,Na-CAPSO pH9.6).Samples were incubated at room temperature with gentle agitation for20min,and then insoluble material was removed by centrifugation at21000×g max for20min
at25°C.The concentration of peptide in each remaining superna-tant was determined by measuring its OD280nm.
To determine the maximum solubility of each peptide,the peptides were dissolved to saturation in peptide buffer by extensive vortexing and incubation with stirring at25°C overnight.Insoluble material was removed by centrifugation at15000×g for15min at25°C and the amount of dissolved peptide was measured directly by amino acid analysis.
Peptide Competitive Displacement of Bound Recombinant PrA from IgG-Sepharose.Recombinant PrA(280µg;6.7nmol) from S.aureus(Pierce)was dissolved in1mL TB-T[20mM HEPES-KOH pH7.4,110mM KOAc,2mM MgCl2,0.1%Tween-20(vol/vol)]and added to280µL of packed pre-equilibrated Sepharose4B(Amersham Biosciences)conjugated with affinity-purified rabbit IgG(ICN/Cappel; 1.87nmoles IgG).After incubation on a rotating wheel overnight at4°C,the resin was washed twice with1mL TB-T,twice with1mL TB-T containing 200mM MgCl2,and twice with1mL TB-T.After the final wash, the resin was divided evenly into14equal aliquots.The peptide was dissolved in peptide buffer at concentrations ranging between0and440µM peptide.Aliquots of400µL of the appropriate peptide solution was added to each PrA-IgG-Sepharose containing tube,and the tubes were then incubated on a rotating platform for3h at4°C followed by1h at25°C. After displacement of bo
und PrA from the IgG-Sepharose,the resin was recovered by centrifugation on a Bio-Spin column (BioRad),and resuspended in one-bed volume of sample buffer.Samples were separated by SDS-PAGE.
Yeast Strains.Strains are isogenic to DH5alpha unless otherwise specified.All yeast strains were constructed using standard genetic techniques.C-terminal genomically tagged strains were generated using the PCR method previously described.7,17
Affinity Purification of Proteins and Protein Complexes on IgG-Sepharose.The protocol for the purification of PrA-containing complexes was modified from published methods.18-20For the purification of Kap95p-PrA,yeast cytosol was prepared essentially as previously described.21,22Kap95p-PrA cytosol was diluted with3.75volumes of extraction buffer 1[EB1:20mM Hepes/KOH,pH7.4,0.1%(vol/vol)Tween-20, 1mM EDTA,1mM DTT,4µg/mL pepstatin,0.2mg/mL PMSF]. The diluted cytosol was cleared by centrifugation at2000×g av for10min in a Sorvall T6000D tabletop centrifuge and at 181000×g max for1h in a Type80Ti Beckman rotor at4°C.10µL bed volume of IgG-Sepharose pre-equilibrated in EB1was added per0.5mL of cytosol and the binding reaction was incubated overnight at4°C on a rotating wheel.The resin was recovered by centrifugation at2000×g av for1min in a Sorvall T6000D tabletop centrifuge,transferred to1.5mL snap-cap tubes(Eppendorf),and w
ashed6times with EB1without DTT. For the purification of Nup82p-PrA,cells were grown in Whickerham’s medium21to a concentration of4×107cells/ mL,washed with water and with20mM Hepes/KOH pH7.4, 1.2%PVP(weight/vol),4µg/mL pepstatin,0.2mg/mL PMSF, and frozen in liquid N2before being ground with a motorized grinder(Retsch).Ground cell powder(1g)was thawed into10 mL of extraction buffer2[EB2;20mM Na-HEPES,pH7.4,0.5% TritonX-100(vol/vol),75mM NaCl,1mM DTT,4µg/mL pepstatin,0.2mg/mL PMSF].Cell lysates were homogenized by extensive vortexing at25°C followed by the use of a Polytron for25s(PT10/35;Brinkman Instruments)at4°C.Clearing of the homogenate,binding to IgG-Sepharose,resin recovery and washing was done as above except that10µL of IgG-Sepharose bed volume was used per1g of cell powder and EB2without DTT was used for all the washes.Elution of the PrA tagged complexes was performed as described below.
Peptide Elution of Test Proteins and Protein Complexes and Removal of Peptide by Size Exclusion.Kap95p-PrA or Nup82p-PrA bound IgG-Sepharose resin was recovered over a pre-equilibrated Bio-Spin column(BioRad)by centrifugation for1min at1000×g max.Three bed-volumes of440µM(unless otherwise indicated in the text)of eluting peptide in peptide buffer were added per volume of packed IgG Sepharose resin. The elution was carried out for various times(as indicated in the text)at either4°C or at25°C.When elution was complete, the eluate was recovered over a Bio-Spin
column.Finally,the resin was washed with one bed-volume of elution buffer to displace more eluted material from the resin and the wash was pooled with the initial eluate.The peptide was removed by filtration of the eluate over a micro spin G25column(Amer-sham Biosciences)as described by the manufacturer.
Kap95p-Nup2p in Vitro Binding Experiments.To demon-strate in vitro binding of proteins after elution from the resin, Kap95p-PrA from0.3mL of yeast cytosol was affinity-purified on17.5µL of packed IgG-Sepharose and eluted with52.5µL of440µM Bio-Ox for2.5h at4°C followed by1h at25°C. The resulting sample(total volume88µL)was mixed with0.1µL li total cell lysate containing Nup2p-GST(generous gift from David Dilworth and John Aitchison23)and brought to a total volume of500µL with TB-T,1mM DTT,4µg/mL pepstatin,0.2mg/mL PMSF.Controls were set up in the absence of either Kap95p-PrA or Nup2p-GST.The samples were incubated at25°C for30min after which40µL of packed,pre-
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equilibrated glutathione-Sepharose 4B resin (Amersham Bio-sciences)was added per sample and the i
ncubation was continued at 4°C for 1h.After nine washes with 1mL of TB-T,1mM DTT,4µg/mL pepstatin,0.2mg/mL PMSF,at 25°C,the resin was recovered on Bio-Spin columns as described above and bound material was eluted with 40µL of sample buffer.The samples were resolved on SDS-PAGE alongside an aliquot of input peptide-eluted Kap95p-PrA.
To demonstrate the recovery of in vitro reconstituted protein complexes from the resin,Kap95p-PrA from 0.3mL of yeast cytosol was affinity-purified on 10µL of packed IgG-Sepharose and the washed resin was equilibrated in TB-T,1mM DTT,4
µg/mL pepstatin,0.2mg/mL PMSF.This pre-bound Kap95p-PrA was mixed with 50µL li total cell lysate containing Nup2p-GST in a total volume of 1mL of TB-T,1mM DTT,4µg/mL pepstatin,0.2mg/mL PMSF.A mock control experiment was set up in the absence of Nup2p-GST.The binding reaction was carried out for 1h at 4°C and the resin was washed 2times with 1mL of TB-T,2times with 1mL of TB-T containing 100µM ATP and 3times with peptide buffer (all washed were without DTT).Bound material was eluted with 30µL of 440µM Bio-Ox in peptide buffer at 4°C for 2.5h at 4°C followed by 1h at 25°C.Samples were resolved by SDS-PAGE.
Figure 1.Addition of a Biotin moiety to the FcIII peptide does not alter the ability of the peptide to comp
etitively displace bound PrA from IgG-Sepharose.(a)Primary sequence and chemical structure of the biotinylated FcIII peptide,Bio-Ox.(b)220µM suspensions of peptides were prepared in buffers of different pHs,and allowed to solubilize.The material remaining in the buffer after centrifugation is plotted for Bio-Ox (closed triangles,black trend line )and FcIII (open circles,gray trend line ;dashed horizontal line represents the starting 220µM level .(c)Increasing amounts of Bio-Ox (closed triangles )and FcIII (open diamonds )were used to competitively displace recombinant PrA from IgG-Sepharose.The amounts of PrA left on the resin after elution were resolved by SDS-PAGE alongside known amounts of PrA standards.The data are displayed on logarithmic scale on both axes.Data are displayed as a %recovery relative to the input PrA amount (i.e.,PrA amount remaining bound in the absence of eluting peptide).Linear regression for both data sets was used to calculate the IC50.
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Quantitation and Image Analyses.Band intensities were quantified with the Openlab software (Improvision),and the data was plotted using Excel (Microsoft).
Results and Discussion
Design of the PrA Mimicking Peptide.The hinge region on the Fc fragment of immunoglobulin G (IgG)interacts with Staphylococcus aureus Protein A (PrA).This region was also found to be the preferred binding site for peptides selected by bacteriophage display from a random library.16The specific Fc binding interactions of a selected 13amino acid peptide (termed FcIII),were shown to closely mimic those of natural Fc binding partners.We reasoned that this peptide could be used to efficiently displace PrA tagged proteins from IgG-
conjugated affinity resins.Initial trials with FcIII determined that,although it functioned as an eluant,it exhibited a strong tendency to aggregate and its solubility under physiological conditions was not sufficient for many practical purposes,leading to low yields and nonreproducible results.As the high peptide concentrations needed for elution are outside the conditions for which the FcIII peptide was designed,we synthesized several modified peptides based on FcIII,with the specific aim of increasing t
heir solubility and decreasing their degree of aggregation under conditions that would be useful for the isolation of proteins and protein complexes.Among the different alternatives,the most efficient in the displacement of bound PrA-tagged Kap95p from IgG-Sepharose was a peptide in which the amino-terminus of the original FcIII peptide was
Figure 2.Bio-Ox can be used to efficiently compete bound PrA-tagged proteins and protein complexes from IgG-Sepharose in a temperature-dependent fashion.(a )Kap95p-PrA/Kap60p was affinity-purified on IgG-Sepharose from logarithmically growing yeast cells.440µM Bio-Ox was used to competitively displace the bound tagged proteins from the IgG-Sepharose resin.The elution reaction was carried out for the times indicated.At the end of the incubation time eluted proteins (E )and proteins remaining bound to the resin (B )were resolved on SDS-PAGE.(b )Kap95p-PrA (closed squares)and Nup82p-PrA (open squares )were affinity-purified on IgG-Sepharose from logarithmically growing yeast cells and eluted as described above.The amounts of eluted versus resin-bound protein was quantified using the OpenLab software and the elution efficiency for each time point is presented as the percentage of eluted material over the total amount of bound plus eluted material (%eluted).(c )440µM Bio-Ox was used to elute Kap95p-PrA or Nup82p-PrA for 1h at 4°C or 25°C as indicated.
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modified by the addition of a Biotin moiety (data not shown).We termed this peptide Bio-Ox (Figure 1,panel a).The solubility of Bio-Ox was measured directly by amino acid analysis and was shown to be ∼3-fold greater than the solubility of FcIII at pH 7.4.In addition,comparison of the solubility of both peptides over a range of pHs indicated that the Bio-Ox was considerably more soluble than FcIII at all but the most extreme pHs tested;importantly,Bio-Ox is very soluble across the full physiological range of pHs (Figure 1,panel b).
To determine whether the addition of the Biotin moiety could have altered the inhibiting ability of the peptide,we measured the inhibition constant for Bio-Ox and found it to be comparable with the reported K i for FcIII (∼11nM;data not shown).We then measured the IC 50for competitive displacement for FcIII and Bio-Ox,under conditions in which both were soluble.For this test,commercially available recom-
binant PrA from S.aureus was first bound to IgG-Sepharose and then increasing concentrations of the peptide were used to displace the bound PrA from the immobilized IgG (Figure 1,panel c).The apparent IC 50was found to be 10.4(3.2µM for FcIII and 9.8(2.6µM for Bio-Ox (mean value of four inde
pendent trials (standard deviation of the mean).Taken together,Bio-Ox appears to be as efficient as FcIII at binding to the F c portion of antibodies and competing for this site with Protein A,but is far more soluble in physiologically compatible buffers,a key requirement for an efficient elution peptide (Figure 3).
Experimental Design of the Competitive Elution Procedure.The principle of the method is as follows;genomically PrA-tagged proteins of interest are expressed in yeast and affinity isolated on IgG-conjugated Sepharose resin.Depending on the conditions used for lysis and extraction,the test protein can be recovered in native form either in isolation or in complexes with protein partners.After binding,the resin is recovered by centrifugation and washed extensively to remove unbound material.The bound material is competitively displaced from the IgG-Sepharose resin by incubation with 440µM Bio-Ox peptide in peptide buffer for 2h at 4°C.Finally,the peptide is rapidly (<1min)removed from the eluted sample by fraction-ation over a size exclusion spin column.Given a typical protein of average abundance,1-10µg of pure protein can be recovered from 1g of cells using this method.
Figure 3.Elution of Kap95-PrA/Kap60p is dose dependent.(a )Kap95p-PrA was affinity-purified on IgG-Sepharose from loga-rithmically growing yeast cells and eluted using increasing concentrations of Bio-
Ox peptide as indicated.(b )The elution efficiency measured as described in Figure 2was plotted versus the peptide concentration in logarithmic scale as indicated.
Figure 4.Eluted Kap95p-PrA/Kap60complex retains its biological activity.(a )Kap95p-PrA was prepared by affinity purification followed by Bio-Ox peptide elution (Kap95-PrA eluate ).Three binding reactions were then set up containing eluted Kap95p-PrA and Nup2p-GST bacterial lysate,Kap95p-PrA alone or Nup2p-GST alone.At the end of the incubation,Nup2p-GST was affinity-purified on glutathione-Sepharose and the immobilized material was eluted from the resin with sample buffer and resolved on SDS -PAGE (GST bound ).(b )Kap95p-PrA was immobilized on IgG-Sepharose and incubated with (+)or without (-)bacterial lysate containing Nup2p-GST.The resulting material was eluted using Bio-Ox.Eluate (E )and resin bound (B )material was resolved on SDS-PAGE.*,indicates a Nup2p breakdown product.Table 1.Elution Efficiency for PrA Tagged Nups
name of nup
%yield
Nup53p 56Nup59p 81Nup84p 88Nup85p 81Nic96p 76Nsp1p 99Nup1p 99Nup120p 69Nup157p 82Nup159p 53Nup170p 80Nup192p 76Gle2p 90
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