One-step purification of bacterially expressed recombinant transducin α-subunit and isotopically...

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Protein Expression and Purification 51 (2007) 187–197

One-step purification of bacterially expressed recombinant transducina-subunit and isotopically labeled PDE6 c-subunit for NMR analysis q

Lian-Wang Guo a,*, Fariba M. Assadi-Porter b, Jennifer E. Grant a,1, Hai Wu a,John L. Markley b, Arnold E. Ruoho a

a Department of Pharmacology, University of Wisconsin, Madison, WI 53706, USAb National Magnetic Resonance Facility at Madison, Department of Biochemistry, University of Wisconsin, Madison, WI 53706, USA

Received 30 May 2006, and in revised form 22 June 2006Available online 22 July 2006

Abstract

Interactions between the transducin a-subunit (Gat) and the cGMP phosphodiesterase c-subunit (PDEc) are critical not only for turn-on but also turn-off of vertebrate visual signal transduction. Elucidation of the signaling mechanisms dominated by these interactions hasbeen restrained by the lack of atomic structures for full-length Gat/PDEc complexes, in particular, the signaling-state complex represent-ed by GatÆGTPcS/PDEc. As a preliminary step in our effort for NMR structural analysis of Gat/PDEc interactions, we have developedefficient protocols for the large-scale production of recombinant Gat (rGat) and homogeneous and functional isotopically labeled PDEcfrom Escherichia coli cells. One-step purification of rGat was achieved through cobalt affinity chromatography in the presence of glyc-erol, which effectively removed the molecular chaperone DnaK that otherwise persistently co-purified with rGat. The purified rGat wasfound to be functional in GTPcS/GDP exchange upon activation of rhodopsin and was used to form a signaling-state complex withlabeled PDEc, rGatÆGTPcS/[U-13C,15N]PDEc. The labeled PDEc sample yielded a well-resolved 1H–15N HSQC spectrum. The methodsdescribed here for large-scale production of homogeneous and functional rGat and isotope-labeled PDEc should support further NMRstructural analysis of the rGat/PDEc complexes. In addition, our protocol for removing the co-purifying DnaK contaminant may be ofgeneral utility in purifying E. coli-expressed recombinant proteins.� 2006 Elsevier Inc. All rights reserved.

Keywords: Recombinant transducin a-subunit; One-step purification; DnaK; PDE6 c-subunit; Isotope-labeled protein; NMR; HSQC

The propagation as well as termination of vertebratevisual signal transduction is dependent on the interactions

1046-5928/$ - see front matter � 2006 Elsevier Inc. All rights reserved.

doi:10.1016/j.pep.2006.07.012

q This work was supported by NIH Grants GM33138 (to AER) andRR02301 (to J.L.M.), and by an Applied Research Grant (to F.M.A.-P.).This study made use of the National Magnetic Resonance Facility atMadison, which is supported by National Institutes of Health GrantsP41RR02301 (Biomedical Research Technology Program, National Cen-ter for Research Resources) and P41GM66326 (National Institute ofGeneral Medical Sciences). Equipment in the facility was purchased withfunds from the University of Wisconsin, the National Institutes of Health(P41GM66326, P41RR02301, RR02781, and RR08438), the NationalScience Foundation (DMB-8415048, OIA-9977486, and BIR-9214394),and the US Department of Agriculture.

* Corresponding author. Fax: +1 608 262 1257.E-mail address: [email protected] (L.-W. Guo).

1 Present address: Department of Biochemistry, University of Medicineand Dentistry of New Jersey-Medical School, Newark, NJ 07103, USA.

between transducin and its effector, the cGMP phosphodi-esterase (PDE6)2 c-subunit (PDEc). Light-excited rhodop-sin catalyzes the exchange of GTP for GDP in thetransducin a-subunit (Gat) thus converting it into an activeconformation that can displace the inhibitory PDEc sub-

2 Abbreviations used: PDE6, rod photoreceptor cGMP phosphodiester-ase; PDEc, PDE6 inhibitory subunit; Gat, transducin a-subunit; rGat, therecombinant Gat used in this study, which is the Chi8 construct of thechimeric Gat/i1; GAP, GTPase activating protein; DnaK, the bacterialhomolog of heat shock protein 70; FPLC, fast performance liquidchromatography; HPLC, high performance liquid chromatography; Co–NTA, Co2+–nitrilotriacetic acid-agarose resin; ESI-MS, electro-sprayionization mass spectrometry; PAGE, polyacrylamide gel electrophoresis;NMR, nuclear magnetic resonance; HSQC, heteronuclear single quantumcoherence; b-ME, b-mercaptoethanol; DTT, dithiothreitol; D2O, deuter-ated water.

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188 L.-W. Guo et al. / Protein Expression and Purification 51 (2007) 187–197

unit from PDE6. This then triggers amplification of visualsignal transduction through activation of PDE6, whichlowers the cGMP level thus causing the closure ofcGMP-gated channels and hyperpolarization of the cellmembrane. Then, the PDEc molecules displaced fromPDE6 are recruited into a GTPase accelerating protein(GAP) complex to rapidly terminate signal transduction(for review, see [1,2]).

Because transducin-dominated visual signaling serves asa paradigm for understanding other G-protein signalingsystems, interactions between Gat and PDEc have beenthe subject of numerous studies. The only high-resolutionstructural model for a Gat/PDEc complex is the crystalstructure of a transition-state GAP complex, which con-tains the recombinant Gat in a GDPÆA1F4

�-bound form,the C-terminal half of PDEc, and the catalytic core ofthe regulator of G-protein signaling RGS9 [3]. The struc-ture of this complex was a major breakthrough in elucidat-ing the nature of Gat/PDEc interactions. However,because in this structure PDEc is truncated and Gat is inthe GDPÆA1F4

�-bound form that mimics the transitionstate, critical information regarding the signaling-statecomplex, represented by GatÆGTPcS/PDEc, is still missing.Given the failure of efforts to solve crystal structure of theGat/PDEc complex with full-length molecules [3], we havedecided to use solution-state NMR spectroscopy to studyPDEc/Gat interactions. Because the PDEc/Gat complexis known to form readily in solution [4–8], this appears tobe a feasible approach. Encouragement comes also fromrecent NMR studies of transducin/rhodopsin interactionsusing bacterially expressed and isotope-labeled recombi-nant Gat [9–11].

Functional recombinant Gat has been heterologouslyexpressed in the Sf9/baculovirus system [12,13], yet nostructural determination has been reported using theseGat constructs, likely due to the limited amount of pureprotein produced by this system. Expression of Gat inEscherichia coli was unsuccessful prior to the generationof chimeric Gat/i1 constructs, in which the Gat a3–b5sequence was substituted by the corresponding region inGai1 [5]. Since then, the production of Gat/i1 by E. coli cellshas facilitated important studies, such as crystal structuresof the transducin heterotrimer [14] and the partial GAPcomplex [3], NMR studies of isotope-labeled Gat/i1

[9–11], and an increasing number of biochemical studies[6,7,15–17].

However, with the original protocol for producing Gat/i1

[5], the protein expression level was relatively low withmuch of the product in inclusion bodies. Moreover, theco-purification of contaminants with the target proteinmandated tedious purification steps: first a nickel affinitycolumn and then an ion exchange column. The recent useof a subtilisin prodomain-Gat/i1 fusion construct led to asignificant improvement in Gat/i1 purification [9].

We report here, a method for producing soluble Gat/i1

protein from E. coli in high yield and with a simplified puri-fication scheme that does not require a new fusion con-

struct. Purification is accomplished in a single step on acobalt column by supplementing the buffer with glycerol.We also describe the economical production of stable-iso-tope labeled PDEc as required for NMR analysis. Datafrom initial 1H–15N HSQC (heteronuclear single quantumcoherence) spectra of [U-15N]PDEc indicate that NMRshould provide a practical approach to the study of Gat/PDEc interactions.

Materials and methods

Reagents and buffers

GTPcS, GDP, ATP, NaF, AlCl3, imidazole, glycerol, b-mercaptoethanol (b-ME), phenyl methyl sulfonyl fluoride(PMSF), acetonitrile, trifluoroacetic acid (TFA), andmolecular weight standards (MW std) were purchasedfrom Sigma–Aldrich. Sequencing-grade modified trypsin(19,400 U/mg, 0.5 mg/ml) was from Promega. [U-13C]glu-cose, [U-15N]NH4Cl, and deuterium oxide (D2O) werefrom Cambridge Isotope Laboratories. Bacto-tryptoneand yeast extract for the LB medium were from Difco. Iso-propyl-1-thio-b-D-galactopyranoside (IPTG) and dithio-threitol (DTT) were from Gold BioTechnology. Co–NTA(Co2+–nitrilotriacetic acid) resin was prepared by strippingthe Ni–NTA Superflow resin (Novagen) with EDTA andthen recharging it with 50 mM CoCl2. Rabbit polyclonalanti-Gat (N-terminus) and anti-PDEc (N-terminus) wereproducts of Affinity Bioreagents. Mouse monoclonalanti-DnaK was from Stressgen.

Buffer TSG: 20 mM Tris–HCl, pH 7.9, 0.5 M NaCl,5 mM MgCl2, 50 lM GDP, 0.2 mM PMSF, 2 mM b-ME.Buffer GAT: 20 mM Tris–HCl, pH 7.9, 0.5 M NaCl,20 mM imidazole, 20% glycerol, 10 mM MgCl2, 30 mMKCl, 2 mM b-ME, 0.2 mM PMSF. Buffer MGD: 15 mMTris–HCl, pH 7.9, 120 mM NaCl, 5 mM MgCl2, 50 lMGDP, 1 mM DTT. Buffer UB: 10 mM HEPES, pH 7.5,120 mM NaCl, 5 mM MgCl2, 5· M9 salt (no NH4Cl):34 g Na2HPO4, 15 g KH2PO4, 2.5 g NaCl in 1 L of H2O.Buffer PES: 50 mM Na2HPO4, pH 7.5, 0.3 M NaCl,1 mM EDTA, 0.2 mM PMSF.

Escherichia coli expression and one-step purification of rGat

The plasmid pHis6-Chi8 for expression of chimericGat/i1 in E. coli [5] was kindly provided by Dr. NikolaiP. Skiba (Duke University). We chose Chi8 because amongsoluble Gat/i1 constructs it has the highest affinity forPDEc and the smallest insertion of Gai1 sequence (residues215–228 and 236–295). For clarity we denote Chi8 by‘‘rGat’’ (recombinant Gat) throughout this paper. Usingthis construct as a template, two new mutants were gener-ated by the QuikChange method (Strategene): VLD, whichhas Val, Leu, and Asp substitutions at the Gat C-terminalpositions 340, 341, and 343, respectively; and K341L,which contains a single mutation with lysine replaced byleucine. C-terminal Gat peptides with these substitutions

L.-W. Guo et al. / Protein Expression and Purification 51 (2007) 187–197 189

were reported to have much higher affinity for light-activat-ed rhodopsin than the corresponding peptide withwild-type sequence [18].

Plasmid pHis6-Chi8 was transformed into E. coli BL21(DE3) cells (Novagen). A 200-ml starter LB culture (10 gtryptone, 5 g yeast extract, and 10 g NaCl per liter) wasinoculated with a single colony, grown at 37 �C overnight,and then transferred to a 3-L LB medium supplementedwith 2 g glucose. The culture was divided into eight 2-Lbaffled-bottom flasks, and grown at 33 �C to an optical celldensity of 0.8–1.0 OD600, 30 lM IPTG was then added forinduction. The induction was continued at 15 �C overnightin an Innova 4340 refrigerated incubator shaker (NewBrunswick Scientific). The final cell density was �5.0OD600. The collected cells of �15 g of wet weight wereresuspended in 150 ml Buffer TSG. Each 50-ml cell suspen-sion was sonicated for 5 min (40% output, 50% duty cycle)by using a Sonifier 250 with a micro-tip (VWR Scientific).The crude lysate was then cleared by centrifugation at25,000g for 40 min at 4 �C in a RC-5B centrifuge (Sorvall)with a GSA rotor. The supernatant was adjusted to BufferGAT supplemented with 3 mM ATP, and then loaded by aWaters 650E FPLC system onto a Pharmacia HR 16/5 col-umn packed with 10 ml Co–NTA resin. After loading, thecolumn was washed with Buffer GAT, and the collection of5-ml fractions was initiated. After passage of �200 ml Buff-er GAT, the column was washed with a slightly higherimidazole concentration (30 mM) for another �100 ml. Agradient of 40–200 mM imidazole in 60 min was thenapplied to elute the remaining proteins from the column.The whole process of FPLC was performed at a flow rateof 1 or 2 ml/min and at room temperature with the super-natant and buffers to be loaded onto the column kept onice. Fractions containing highly purified rGat were pooled,supplemented with 50 lM GDP, and then stored at�80 �C. The final yield of P95% pure rGat (estimated byCoomassie staining) was typically >10 mg/L E. coli culture.

The protocol developed here was based on the ‘‘stan-dard’’ recipe, the method published by Skiba et al. [5] (alsosee the instruction from Novagen). The column was regu-larly regenerated with 50 mM MES buffer, pH 5.0. In somecases, the column was stripped with 100 mM EDTA, pH7.5, and then recharged with 50 mM CoCl2 after washingthe column. The purple color of cobalt is a convenientindicator of column regeneration.

GTPcS loading to purified rGatÆGDP

Purified rGatÆGDP was concentrated �10· on an Ami-con concentrator with a 30 kDa cutoff (Millipore), andthen dialyzed overnight at 4 �C against Buffer MGD of500· volume in SPECTRA/POR dialysis tubing with acutoff of 6–8 kDa (Spectrum Medical Industries). One mil-limolar GTPcS and 200 lg rhodopsin in uROS (urea-treat-ed rod outer segment) [19] were added to 2 ml of 0.2 mMrGatÆGDP in dim red light, and incubated for 10 min inthe dark. The mixture contained in microcentrifuge tubes

on ice was then exposed to a 50-W table light at a distanceof 10 cm for 1 min. After a 1-h incubation at 4 �C withoccasional shaking, the mixture was spun at 16,000g and4 �C for 1 h to pellet the uROS membranes. The superna-tant was aliquoted and stored at �80 �C. Alternatively,GTPcS could be loaded by incubation with rGatÆGDP at4 �C overnight in the absence of uROS.

The efficiency of GTPcS loading was assessed by trypsinprotection assay [20]. A standard reaction of 8 ll included1· Buffer UB, 1.5 lg rGat, and 0.015 lg trypsin. The reac-tion was incubated at room temperature for 30 min andterminated by boiling for 5 min immediately after addingsample buffer containing SDS and DTT.

Escherichia coli expression and purification

of [U-13C,15N]-labeled PDEc

A construct for expressing wild-type (wt) PDEc wasgenerated as an intein fusion with chitin binding domain[8]. A mutant (C68A) was then generated to replace the sin-gle cysteine at position 68 in PDEc by alanine [21], and thisconstruct was transformed into E. coli BL21 (DE3) cells forpreparation of [U-13C,15N]-labeled C68A. A starter cultureof 50 ml LB was inoculated with a single colony, grownovernight at 37 �C, and then centrifuged at 1200g at roomtemperature for 5–10 min to collect the cells. The pelletedcells were washed twice with 1· M9 salt (7 g Na2HPO4,3 g KH2PO4, and 0.5 g NaCl per liter of H2O) and thentransferred to 1 L of [U-13C,15N]M9 medium (1· M9 salt,2 mM MgSO4, 0.1 mM CaCl2, 2 g [U-13C]glucose, 1 g[U-15N]NH4Cl), which was divided into two baffled-bot-tom flasks for growth at 37 �C. When the cell densityreached �1.2 OD600, IPTG was added to a final concentra-tion of 0.5 mM, and the growth was continued for an addi-tional 4–5 h at 33 �C. The final cell density was 3.5–4.0 atOD600.

The harvested cells were lysed by sonication in 50 mlBuffer PES supplemented with 0.1% Triton X-100 and thencleared by centrifugation, as described above for rGat. Thesupernatant was mixed with 5 ml chitin beads (New Eng-land Biolabs), incubated at room temperature for 1 h undergentle shaking, and then loaded to a column by gravityflow. After washing with 50–100 ml Buffer PES, the columnwas flushed with 10 ml Buffer PES supplemented with100 mM b-ME, and left overnight at room temperature.PDEc was then collected and the protein estimated to beP90% pure by Coomassie staining after SDS–PAGE(polyacrylamide gel electrophoresis). The overnight b-MEincubation step was repeated twice to recover more PDEc.The PDEc preparation was further purified by two steps ofreversed-phase HPLC (high performance liquid chroma-tography) (Waters 626 LC System) in acetonitrile solutionsat a flow rate of 1 ml/min. Solution A: 0.1 % TFA; solutionB: 90% acetonitrile, 0.1% TFA. The first HPLC purifica-tion was performed on a column packed with POROS 20R2 resin (PerSeptive Biosystems). Gradient: 2–12 min/ 10–25% B fi 12–42 min/25–46% B fi 42–52 min/46–70% B.


PDEc eluted at �30% acetonitrile was lyophilized forstorage, or loaded onto a Vydac C18 column for the secondHPLC purification. Gradient: 2–18 min/20–48% B fi 18–42 min/48–51% B fi 42–52;min/51–60% B fi 52–70 min/60–90% B. The fraction eluting at �44% acetonitrile, whichcontained the PDEc peak, was lyophilized and stored des-iccated at �80 �C. Typically, �1.5 mg >95% pure[U-13C,15N]PDEc was prepared from 1 L of M9 culture.For a preliminary NMR determination, [U � 15N]-labeledwt-PDEc was prepared similarly, with [U-15N]NH4Cl asthe sole nitrogen source in the M9 culture.

Characterization of purified [U-13C,15N]PDEc by mass

spectrometry and PDEc functional assay

Electrospray ionization mass spectrometry (ESI-MS) ofthe HPLC purified [U-13C,15N]PDEc was performed in theMass Spectrometry Facility of the Chemistry Departmentat the University of Wisconsin-Madison. Samples wereanalyzed on a Shimadzu 2010 LC–MS (Columbia, MD)equipped with a C18 column (Supelco Discovery BIO WidePore C18, 5 U, 150 · 2.1 mm, 200 ll/min). Mobile phaseA = 0.4% formic acid/water. Mobile phase B = 0.2%formic acid/acetonitrile. A standard gradient was usedconsisting of 5–95% B.

For PDEc functional assay, transducin GTPase activitywas determined by a single turnover technique [22]. uROSmembranes lacking endogenous activity of RGS9 wereused as a source for the photoexcited rhodopsin requiredfor transducin activation. The reactions were initiated bythe addition of 10 ll of 0.6 lM [U-32P]GTP (�105 dpm/sample) to 20 ll of uROS membranes (20 lM final rhodop-sin concentration) reconstituted with transducin heterotri-mer (1 lM) and recombinant RGS9-Gb5 complex(0.5 lM), either in the absence or presence of PDEc.

Complex preparation using purified [U-13C,15N]PDEc and

rGat

To prepare the rGat/PDEc complex, 2 mg[U-13C,15N]PDEc was first added to 4 ml of Bis–Tris bufferat pH 6.7; 10 mg rGatÆGTPcS in 8 ml of the same bufferwas then slowly added with gentle vortexing. The mixturewas concentrated in an Amicon Ultra Centrifugal FilterDevice (5 kDa cutoff, Millipore) by spinning at 2400g

and 4 �C in an IEC Clinical Centrifuge. The resulting�500 ll sample contained [U-13C,15N]PDEc at aconcentration of �0.4 mM. A complex of GatÆGTPcS/[U-13C,15N]PDEc was prepared similarly. NativeGatÆGTPcS was purified from bovine retinas according tothe established method [20], as also described by Grantet al. [8].

Formation of the rGat/PDEc complex was assessed bynative PAGE assay. A sample of 2 lM rGatÆGTPcS wasprepared in Buffer UB. Then [U-13C,15N]PDEc was addedto achieve various molar ratios (0.2–2.0) vs. rGatÆGTPcS,and the mixtures were incubated on ice for 30 min. The

reactions were mixed with sample buffer devoid of SDSand then subjected to native PAGE (under SDS-free condi-tions) as described previously [23].

NMR spectroscopy of isotope-labeled PDEc

The protocols used for NMR data collection were asdescribed previously [24], except as noted. Briefly, two-di-mensional 1H–15N HSQC [25] data sets were recorded at10 �C on a Bruker DMX600 spectrometer equipped witha triple resonance (1H/13C/15N) Z-axis gradient probe.Water suppression was achieved with a Watergatesequence containing a 3-9-19 selective inversion pulse.Spectra were collected on the [U-15N]-labeled wt-PDEcthat was dissolved in 90% H2O and 10% D2O. The pH ofthe sample measured with a micro-pH electrode was 3.5.

Other methods

Western blot analysis of proteins was performed follow-ing SDS–PAGE using 12% gels as previously described[23]. The proteins were electro-transferred to an Immobi-lon-P PVDF membrane (0.45 lm, Millipore), incubatedwith a primary antibody, and then detected by the horse-radish peroxidase-conjugated secondary antibody usingthe SuperSignal West Dura substrates (Pierce). Dilutionof the primary antibodies was: anti-Gat, 2000-fold; anti-PDEc, 4000-fold; and anti-DnaK, 4000-fold. Determina-tion of protein concentration and quantitation of theCoomassie-stained protein bands on SDS gels wereconducted as previously described [23].

Results and discussion

One-step purification of rGat through Co–NTA affinity

chromatography

Fig. 1A shows the one-step purification profile of the E.coli-expressed His6-tagged rGat through affinity chroma-tography. Instead of using a Ni–NTA column accordingto the standard protocol [5], a Co–NTA column wasemployed in our method with modified buffer conditionsin which 20% glycerol was included (see Materials andmethods). Unlike the standard Ni–NTA chromatography,surprisingly, P90% pure rGat was eluted after washingwith one column volume (�10 ml) of Buffer GAT(Fig. 1A). Therefore, the pure rGat was directly eluted bythe wash buffer containing 20 mM imidazole (Fig. 1A, frac-tions 4–40). With a Ni–NTA column, this low concentra-tion of imidazole usually is used to prevent nonspecificprotein binding, rather than for eluting a His6-tagged tar-get protein. A slightly higher concentration (30 mM) ofimidazole eluted more rGat (fractions 41–60). When theimidazole gradient was applied (200 mM maximum con-centration), only a minor amount of additional rGat waseluted, together with a contaminant protein of 70 kDa(fractions 1 0–20 0). This observation indicates that most of

A

B

Fig. 1. Comparison of the Co–NTA chromatographic purification ofrGat by our one-step protocol and the standard protocol. Presented areCoomassie-stained SDS–PAGE denaturing gels (12%, 18 · 16 · 0.075 cm).The numbers of the rGat fractions loaded to the gels are labeled under thecorresponding lanes. The primed numbers (e.g. 8 0) represent the fractionseluted by the imidazole gradient (40–200 mM in 1 h). (A) One-steppurification of rGat using a Co–NTA column (see Materials and methods)illustrated by three Coomassie-stained SDS gels. The supernatant prior tocolumn loading (PL), flow through (FT), and selected fractions (40 ll each)were loaded. Fractions 1–40 were eluted at 20 mM imidazole, 41–63 at30 mM, 1 0–20 0 by the imidazole gradient. *Fractions 9 and 3 were runagain (on the 2nd and the 3rd gels, respectively), to show that thebackground of these two lanes on the 1st gel was actually due to thespillover from the neighbor lane FT or PL (as also seen in the lane of MWstd). (B) SDS gel showing the Co–NTA purification of rGat performedwith the standard protocol [5], on which a major contaminant of 70 kDais clearly shown. The loaded fractions were eluted by the imidazolegradient.

Table 1Purification of rGat and [U-13C,15N]PDEc

Recombinant proteins Purification steps Proteina (mg) Purityb (%)

rGat Co–NTA column �12.0c P95[U-13C,15N]PDEc Chitin column �2.0 >90

POROS column �1.5 �99

Protein amount in crude lysate was not determined.a Amount of pure protein from 1-L cell culture.b Estimated using Coomassie-stained SDS gels.c Pooled pure rGat fractions eluted from the Co–NTA column.


the pure rGat was recovered from the Co–NTA column bywashing with glycerol-containing Buffer GAT, beforeapplying the final imidazole gradient. A yield of at least10 mg >95% pure rGat (per liter culture) was obtained bypooling the pure fractions 4–40 (Fig. 1A, the 3rd gel, thelast lane) (Table 1).

This one-step purification profile (Fig. 1A) is comparedto that (Fig. 1B) in which rGat was purified using the sameCo–NTA column but with the standard protocol with noglycerol included [5]. Conspicuously, with the standard

protocol a major contaminating protein of 70 kDa co-puri-fied with rGat; both proteins were not eluted until the finalimidazole gradient was applied. This contaminating pro-tein was identified to be DnaK by Western blotting(Fig. 2A). The presence of DnaK together with other con-taminants of lower molecular weights renders a secondarystep of purification inevitable. This comparison shows thatglycerol (and ATP) present in Buffer GAT enabled one-step purification by preventing DnaK from co-purifyingwith rGat.

Glycerol effectively removed contaminating DnaK from rGat

As part of the effort to improve the rGat purificationmethod, we optimized the expression of rGat in E. coli

by induction at a higher cell density (OD600 � 1.0) anda lower temperature (15 �C), which dramaticallyincreased the expression level of soluble rGat (Fig. 2B,lane 3). By contrast, rGat expressed by the standard pro-cedure could barely be seen on the gel (Fig. 2B, lane 2).Slower protein synthesis at the lower temperature likelypromoted proper folding of rGat and thereby preventedinclusion body formation. Because IPTG induction tendsto suppress cell growth, starting induction at a highercell density probably led to a higher number ofprotein-producing cells.

Purification of rGat on a Ni–NTA column according tothe standard protocol [5], resulted in the persistent co-puri-fication of rGat with two major contaminants: a 70-kDaprotein (DnaK) and a 24-kDa protein which was not iden-tified in our study (Fig. 2C, lane 1). The 24-kDa contami-nant protein disappeared after the column was changed toCo–NTA (Fig. 2C, lane 2), whereas DnaK still remainedand could not be removed, even by a secondary purifica-tion step on a Pharmacia Mono Q ion exchange column(Fig. 2C, lane 3). DnaK is the bacterial homolog of themolecular chaperone Hsp70 (heat shock protein) that helpsprotein folding [26] and DNA replication fork repair [27].Because MgATP induces a conformational change inDnaK that lowers its substrate binding affinity [28,29], wefirst tried adding ATP to remove the contaminating DnaK[30,31]. However, inclusion of ATP in the purification buff-er system did not eliminate the co-purification of DnaKwith rGat (Fig. 2C, lane 5), even in the presence of TritonX-100, which was expected to disrupt any possible interac-tion between DnaK and rGat (Fig. 2C, lane 6). It has been

A

C

B

Fig. 2. Optimization of the conditions for E. coli expression andpurification of rGat. (A) Western blotting was performed (Materials andmethods), to confirm that the 70-kDa contaminant protein was DnaK.Lane 2 is the MW std, and the top band is 66 kDa. (B) Expression of rGat

in E. coli following the standard protocol [5] (lane 2) and under theoptimized conditions (Materials and methods) (lane 3): induction initiatedat 1.0 OD600 and continued overnight at 15 �C. The supernatant samplesloaded in lanes 2 and 3 were from approximately the same number ofE. coli cells. (C) Various conditions for Co–NTA column purification ofrGat samples, as visualized by Coomassie staining. Results from thestandard protocol [5] are in lanes 1–7 (shown are the proteins eluted by theimidazole gradient); results from the one-step purification protocol(Materials and methods) are in lanes 8 and 9. Lane 1, Ni–NTA column.Lane 2, Co–NTA column. Lane 3, Co–NTA column then Mono Qcolumn. Lane 4, MW std. Lanes 5–9, various supplements (3 mM ATP,0.1% Triton X-100, 0.5 mg/ml denatured E. coli extract, 20% glycerol)were added into the purification buffer, as indicated above the corre-sponding lanes; a Co–NTA column was used for these conditions.

Fig. 3. Trypsin protection assay of pure rGat loaded with GTPcS orGDPÆAlF4

�. GTPcS loading and trypsin digestion of rGat (and nativeGat) were performed as described in Materials and methods. GDPÆAlF4

�

was loaded by incubating rGat (or Gat) with 50 lM GDP, 30 lM AlCl3,and 10 mM NaF at room temperature for 5–7 min. SDS–PAGE wasperformed with a 15% mini gel (10 · 8 · 0.075 cm).


previously reported that the addition of soluble denaturedE. coli proteins can compete with the target protein forbinding with chaperones such as GroEL and DnaK, whenATP is not sufficient to prevent them from co-purification[32,33]. We also tried this strategy, but DnaK still co-puri-fied with rGat (Fig. 2C, lane 7). Unexpectedly, however,supplementation of MgATP with 20% glycerol removedDnaK almost completely and led to the elution of purerGat (Fig. 2C, lane 8) at a low imidazole concentration,as described above (Fig. 1A). In addition, two rGat

mutants (VLD and K341L, see Materials and methods)

were also readily purified by the glycerol supplement proto-col (data not shown), further proving the effectiveness ofthe one-step purification method.

We then tested the rGat sample purified through ourone-step method for activation by the nonhydrolyzableGTP analogue, GTPcS. It is well documented that Gat

undergoes a conformational change upon GTP/GDPexchange catalyzed by rhodopsin [34,35], and that theactive conformation (represented by GatÆGTPcS) rendersGat resistant to trypsin digestion [20]. Binding of anotherGTP analogue, GDPÆA1F4

�, which confers Gat a confor-mation that mimics the transition state, has the same effect[36]. As with native Gat, rGat samples loaded either withGTPcS or A1F4

� remained as a major 32-kDa Coomas-sie-stained band following trypsin treatment, indicating acorrect activated conformation (Fig. 3).

Therefore, highly pure and properly folded rGat can beproduced in high yield by our protocol for optimizedE. coli expression followed by one-step purification. Theone-step rGat purification method described here has sev-eral obvious advantages over prior approaches. First, theglycerol used in our method did not disrupt the properconformation of rGat, and therefore need not be removedafter purification. Glycerol often is used to protectproteins during storage. Second, glycerol is an inexpensivereagent. Even less glycerol (10%) led to a similar effect inrGat purification (data not shown) although 20% glycerolappeared to be optimal. By using glycerol, it was not nec-essary to use the relatively more expensive ATP in thewash step (see Materials and methods). In fact, with noATP in the whole purification process, we still achievedsimilar results (Fig. 2C, lane 9). The omission of ATPavoids its interference with protein detection by UVabsorbance spectroscopy during FPLC runs. Third,because pure rGat elutes at a low concentration of imid-azole (20 mM or even lower), an extra dialysis step for the

A

B

C

Fig. 4. Preparation of isotopically labeled PDEc. (A) Coomassie-stained16% SDS gel showing the E. coli expression (lanes 1–5) and purification(lanes 6–13) of [U-13C,15N]PDEc (the C68A mutant). The molecularweights of the standards in lane 7 are labeled on the left side of the gel.Loaded in lane 14 is the PDEc-intein fusion protein partially cleaved byDTT. (B and C) The HPLC chromatograms using a POROS column anda C18 column, respectively, which were recorded as UV absorption at dualwavelengths (upper, 215 nm; lower, 280 nm). The residual intein contam-ination (lane 9 in A) after chitin affinity chromatography was readilyseparated from PDEc by the POROS column (see the peaks in B, andlanes 10 and 11 in A). PDEc was further resolved into two peaks by theC18-HPLC in C, which migrated at the same position on the SDS gel(lanes 12 and 13 in A). *Chitin beads-treated [U-15N] wt-PDEc is shown inlane 8 (A) as an example that incubating with a small amount of chitinbeads (30 min at room temperature) can effectively remove the residualintein contaminant (see the intein band, lane 9 in A).


removal of imidazole may be unnecessary. Additionally,we have found, in fact, that rGat can be purified withoutneeding an FPLC system (for programming an imidazolegradient), just through gravity flow elution from theCo–NTA column following the one-step purification pro-tocol (data not shown).

Preparation of highly homogenous and functional

[U-13C,15N]PDEc

A long-term goal of our research is to investigate Gat/PDEc interactions by NMR spectroscopy. We thereforeoptimized a large-scale production of [U-13C,15N]PDEc(here PDEc refers to the C68A mutant) based on the pre-vious protocol for expression and purification of inteinfusion proteins [37]. The intein fusion strategy includesthe potential for producing segmentally labeled PDEc forNMR study through expressed protein ligation [8,38].The cysteine-less construct C68A was used as a precautionto avoid any possible aggregation of wt-PDEc caused byintermolecular disulfide bond formation. Although Cys68is shown to be in close proximity with Gat in the crystalstructure of the partial GAP complex [3], the C68A substi-tution does not affect the interaction of PDEc withGatÆGTPcS [4], as also indicated by the PDEc functionalassay data shown below. This is probably because theproperties of Cys and Ala are not very different. Soluble[U-13C,15N]PDEc-intein fusion protein was produced at ahigh level in the labeled M9 medium (Fig. 4A). Inductionat a higher cell density (OD600 = 1.0–1.2) significantlyimproved the protein yield over that achieved with thecommonly used condition of induction at 0.5 OD600 [37](data not shown), and >90% pure [U-13C,15N]PDEc wasobtained by one-step of chitin affinity chromatography(Fig. 4A, lane 9) (Table 1). Taking the advantage thatPDEc apparently lacks secondary structure, the PDEcsample can be further purified by reversed-phase HPLCin the solvent of acetonitrile. The HPLC-purified PDEcretains its function [8,21,23]. The residual contaminatingintein protein was thus readily removed by reversed-phaseHPLC using a POROS column, and �99% purity of[U-13C,15N]PDEc was achieved as estimated by Coomas-sie-staining (Fig. 4B and lane 11 in 4A). Alternatively, asimilar purity could be gained by simply cleaning up theintein contaminant by adding a small amount of chitinbeads (Fig. 4A, lane 8). A protein sample of this purity isgenerally suitable for NMR analysis.

A further HPLC step on a C18 reversed-phase columnresolved the POROS column-purified [U-13C,15N]PDEcinto two overlapping peaks, which were too close to be sep-arated (Fig. 4C). Therefore both peaks contained a majorpopulation with a measured mass exactly the same as thepredicted (10181, peak a in Fig. 5A and B), and a minorpopulation with a lower mass (Fig. 5A and B, peak b).The mass difference between these two populations is closeto 131, the mass of methionine. This is consistent with theobservation that the N-terminal methionine is commonly

removed during posttranslational processing of proteins[39]. An extra population with a mass adduct of 60(Fig. 5B, peak c) was probably responsible for the shiftof the second peak (peak II) from peak I. Although thismass adduct could not be identified, it did not affect thefunction of PDEc (Fig. 5C). Therefore, these data demon-strate that we were able to produce highly homogeneousand functional isotope-labeled PDEc suitable for NMRstudies.

A

B

C

Fig. 5. Characterization of HPLC-purified [U-13C,15N]PDEc. The twoPDEc peaks resolved by the C18 column (peaks I and II, Fig. 4C) weresubjected to ESI-MS, as shown in (A and B), respectively. The mass of[U-13C,15N]-C68A was predicted to be 10181.17 using PeptideMass ofExPASy (Swiss Institute of Bioinformatics). Each measured mass isexpressed as a mean ± standard deviation, which was calculated bymultiplying the m/z values by charge. The function of purified PDEc forstimulating GTPase activity of transducin (Khydr) was measured asdescribed in Materials and methods, and presented as a mean ± standarddeviation in (C). No functional difference was observed between peak Iand peak II, as indicated by the Khydr values of the two equivalent peaksfrom a PDEc mutant L3C [21], which contains a cysteine in the N-terminus and also an alanine substitution at position 68. These sampleswere prepared using the same protocol for peaks I and II.

A

C

B

Fig. 6. Formation of the complex between [U-13C,15N]PDEc andrGatÆGTPcS. Fifteen percent SDS gel analysis of complexes formedbetween [U-13C,15N]PDEc and (A) native GatÆGTPcS or (B) rGatÆGTPcS(prepared as described in Materials and methods). Different dilutions ofthe [U-13C,15N]PDEc/rGatÆGTPcS complex were loaded onto the gel (B,lanes 6 and 7) to show the molar ratio of rGatÆGTPcS to [U-13C,15N]PDEc(�1.2:1), as estimated by the Coomassie-stained bands. A chitin column-purified sample of [U-13C,15N]PDEc was used as a molecular standard (B,lane 8). The Coomassie-stained native gel in C shows that a complex couldbe readily formed between [U-13C,15N]PDEc and rGatÆGTPcS (lanes 8–13), even at a concentration (�2 lM) much lower than shown in B (�0.4mM). GatÆGTPcS was used as a control to form complex with PDEc (C,lanes 1–6). Molar ratios of PDEc/rGatÆGTPcS (or GatÆGTPcS) are shownat the top of gel. Lane 7 is PDEc alone. Lane 14 is rGatÆGDP.


Signaling-state complex formed by rGatÆGTPcS and

[U-13C,15N]PDEc

Once pure rGat and double labeled PDEc were readilyproduced in large quantity, we were able to further preparethe complex between rGatÆGTPcS and isotope-labeledPDEc. Precipitation was minimized by mixing the two

proteins at a low concentration and then concentratingthe mixture. The rGatÆGTPcS/[U-13C,15N]PDEc complexwas thus obtained at a high concentration (�0.4 mM)(Fig. 6B). Formation of the signaling-state complex ofrGatÆGTPcS/[U-13C,15N]PDEc was confirmed by nativegel assay (Fig. 6C). Similar to native Gat, rGatÆGTPcSmigrated as one major band in the native gel, indicatinga single folded species. PDEc alone was not seen on thenative gel, likely because of its high isoelectric point(9.52, calculated by ExPASy). It is possible that the PDEcmolecules were positively charged in the gel running bufferof pH 8.0 and thereby did not migrate into the gel. Afteradding PDEc to Gat, while the Gat band on the nativegel was reduced a new band at a higher position appearedand increased with more PDEc added; the same was truefor rGat (Fig. 6C). Since an increase either in positive char-ge or molecular size could reduce the mobility of a proteinin a native gel, the up-shifted band is a strong indicationthat PDEc which was positively charged formed complexwith GatÆGTPcS (or rGatÆGTPcS) thus slowed down itsmigration.


NMR spectroscopy of PDEc

The 1H–15N HSQC spectrum (Fig. 7) represents thefirst reported NMR analysis of PDEc. In a 1H–15NHSQC spectrum, cross-peaks correspond to directly bond-ed proton-nitrogen pairs within the protein. One peak isexpected from each residue (except two from glycine, nonefrom proline, and none from the N-terminal residue). The76 isolated cross-peaks observed in the PDEc 1H–15NHSQC spectrum (Fig. 7B) account for the full-length87-amino acid PDEc molecule, because 10 Pro residuesin PDEc (Fig. 7A) are not detectable. The only Trp, thatat position 70 in the PDEc C terminus, is clearly recogniz-able (Fig. 7B). This residue is critically important for thePDEc interaction with Gat [3,4,40]. Generally, dispersedpeaks indicate proper protein folding, whereas unfoldedproteins tend to have peaks confined to a 1H chemicalshift range of 7.5–8.5 ppm and a 15N range of 110–

A

B

Fig. 7. 1H–15N HSQC spectrum of PDEc. (A) Amino acid sequence of the bo[U-15N]PDEc. The HPLC-purified and lyophilized PDEc sample was re-dissoland the pH was 3.5. The 1H–15N HSQC spectrum was recorded as described indenoted by n.p. (negative peak) have negative intensity; they correspond to ly

125 ppm [41]. Most of the PDEc 1H–15N cross-peaks fallin this central region (Fig. 7B), suggesting that PDEcalone is not well structured. This is consistent with ourcircular dichroism measurement of PDEc (data notshown). Thus, PDEc is categorized as an ‘‘intrinsicallyunstructured protein’’ [42,43]. On the other hand, a smallpart of the C-terminal half of PDEc (corresponding to�10 peaks outside the center of the HSQC spectrum,see Fig. 7B) appears to be partially structured. This obser-vation is consistent with NMR relaxation data for[U-13C,15N]PDEc (unpublished data, F.M. Assadi-Porter)that suggest the presence of three possible structuralelements in the C-terminal half of PDEc; these do notoverlap significantly with the three a-helices in the partialGAP structure [3]. An ‘‘intrinsically unstructured protein’’usually adopts a structure when binding to its interactionpartner and thereby has the flexibility to interact withmultiple signaling targets [43].

vine rod wt-PDEc [47]. (B) Two-dimensional 1H–15N HSQC spectrum ofved in H2O containing 10% D2O; the final concentration was �0.25 mM,Materials and methods. The 1H–15N cross-peak of Trp70 is labeled. Peakssine side chains located outside the spectral region and folded in.


It will be therefore of interest to use NMR analysis toexplore the functional nature of PDEc by comparing itsstructural properties when free in solution and when com-plexed with Gat. A Gat/i1 mutant with two Gat residues(His244 and Asn247) substituted back into the Gai1

sequence, which confers it an affinity with PDEc nearly ashigh as native Gat [6], is proposed to be the optimum con-struct for our ongoing efforts in the NMR analysis ofPDEc/Gat interactions (Nikolai O. Artemyev, personalcommunication). Given the critical role of PDEc in the visu-al system [40,44–46] and its functions in nonretinal tissues[47,48], and the fact that the only atomic structure of PDEcis from the C-terminal half in the partial GAP complex [3],structural information gained by NMR on full-lengthPDEc, either free or in a complex, should provide importantinsights into the mechanisms of PDEc-regulated pathways.

Conclusion

We have presented efficient protocols for bacterial pro-duction of large amounts of homogeneous and functionalrGat and stable-isotope labeled PDEc. The addition ofglycerol to the purification buffer provided the key one-steppurification of rGat by effectively removing the major con-taminant, DnaK. We obtained a high quality 1H–15NHSQC NMR spectrum of labeled PDEc and demonstratedthat a PDEc/rGat complex could be prepared with expect-ed functional properties. Future NMR studies of PDEc/Gat interactions thus appear practical. In addition, themethod we developed for removing DnaK, a common con-taminant of proteins produced in E. coli, may be applicableto the purification of other recombinant proteins.

Acknowledgments

We thank K.A. Martemyanov who worked in the labo-ratory of V.Y. Arshavsky at Harvard Medical School forthe functional assay of PDEc. We also thank M.M. Ves-tling at the University of Wisconsin for performing ESI-MS and data processing. Thanks also go to M. Arbabianfor preparing a portion of the Gat samples, and M.K. Sie-vert and D. Thiriot for sharing helpful ideas.

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