Purification and characterization of recombinant ligand-binding domains from the ecdysone receptors of four pest insects

Protein Expression and PuriWcation 53 (2007) 309–324 www.elsevier.com/locate/yprep

PuriWcation and characterization of recombinant ligand-binding domains from the ecdysone receptors of four pest insects Lloyd D. Graham a,¤, Patricia A. Pilling b, Ruth E. Eaton a,1, JeVrey J. Gorman b,2, Carl Braybrook c, Garry N. Hannan a, Anna Pawlak-Skrzecz a, Leonie Noyce a,3, George O. Lovrecz b, Louis Lu b, Ronald J. Hill a a b

CSIRO Molecular and Health Technologies, Sydney Laboratory, P.O. Box 184, North Ryde, NSW 1670, Australia CSIRO Molecular and Health Technologies, Parkville Laboratory, 343 Royal Parade, Parkville, Vic. 3052, Australia c CSIRO Molecular and Health Technologies, Ian Wark Laboratory, Bag 10, Clayton South, Vic. 3169, Australia Received 31 October 2006, and in revised form 15 December 2006 Available online 24 December 2006

Abstract Cloned EcR and USP cDNAs encoding the ecdysone receptors of four insect pests (Lucilia cuprina, Myzus persicae, Bemisia tabaci, Helicoverpa armigera) were manipulated to allow the co-expression of their ligand binding domains (LBDs) in insect cells using a baculovirus vector. Recombinant DE/F segment pairs (and additionally, for H. armigera, an E/F segment pair) from the EcR and USP proteins associated spontaneously with high aYnity to form heterodimers that avidly bound an ecdysteroid ligand. This shows that neither ligand nor D-regions are essential for the formation of tightly associated and functional LBD heterodimers. Expression levels ranged up to 16.6 mg of functional apo-LBD (i.e., unliganded LBD) heterodimer per liter of recombinant insect cell culture. Each recombinant heterodimer was aYnity-puriWed via an oligo-histidine tag at the N-terminus of the EcR subunit, and could be puriWed further by ion exchange and/or gel Wltration chromatography. The apo-LBD heterodimers appeared to be more easily inactivated than their ligand-containing counterparts: after puriWcation, populations of the former were <40% active, whereas for the latter >70% could be obtained as the ligand–LBD heterodimer complex. Interestingly, we found that the amount of ligand bound by recombinant LBD heterodimer preparations could be enhanced by the non-denaturing detergent CHAPS (3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate). Purity, integrity, size and charge data are reported for the recombinant proteins under native and denaturing conditions. Certain intra- and intermolecular disulWde bonds were observed to form in the absence of reducing agents, and thiol-speciWc alkylation was shown to suppress this phenomenon but to introduce microheterogeneity. Crown copyright © 2006 Published by Elsevier Inc. All rights reserved. Keywords: Ligand-binding domains; Ecdysone; Ecdysteroid; Ponasterone A; Receptor; PuriWcation; Characterization; DisulWde bond; Alkylation; Ligand stoichiometry; CHAPS

The ecdysone receptor is a nuclear hormone receptor found in arthropod cells. The molting, metamorphosis and reproduction of insects are controlled by the binding of 20-

*

Corresponding author. Fax: +61 2 9490 5010. E-mail address: [email protected] (L.D. Graham). 1 Present address: Westmead Centre for Oral Health, Westmead Hospital, P.O. Box 533, Wentworthville, NSW 2145, Australia. 2 Present address: Queensland Institute of Medical Research, Post OYce, Royal Brisbane Hospital, Herston, Qld 4029, Australia. 3 Present address: 18 Marrickville Avenue, Marrickville, NSW 2204, Australia.

hydroxyecdysone to this ligand-activated transcription factor [1]. SigniWcant eVort has been made to purify naturally occurring ecdysone receptors, but the isolation and puriWcation of these proteins from insect tissues has been confounded by their low abundance and labile nature [2–5]. Fortunately, cDNAs for the ecdysone receptor protein (EcR)4 and its partner, the ultraspiracle protein (USP), have now been 4 Abbreviations used: LBDs, ligand binding domains; CHAPS, 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate; EcR, ecdysone receptor protein; USP, ultraspiracle protein; PMSF, phenylmethanesulfonyl Xuoride; IMAC, immobilized metal aYnity chromatography.

1046-5928/$ - see front matter Crown copyright © 2006 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2006.12.011

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cloned from a number of organisms. Both belong to the nuclear receptor superfamily and exhibit the domain architecture (A/BCDE/F) characteristic of this group [6,7]. For both proteins, the amino acid sequences of the C- (DNAbinding) and E- (ligand-binding) domains are the most highly conserved. It is now known that EcR and USP form a heterodimer which functions as the natural ecdysone receptor [8]. Although USP is an essential partner for the highaYnity binding of ecdysteroids, the ligand-binding pocket resides in the EcR protein. Full-length or truncated EcR and USP proteins from Drosophila melanogaster and other dipterans have been expressed recombinantly, mostly by coexpression in bacteria, with a view to puriWcation [9–14]. The same is true of some lepidopteran EcR and USP proteins [15,16]. Recently, the atomic interactions between ligands and the ligand-binding domains (LBDs) of recombinant EcR–USP complexes from diVerent insect orders have been elucidated by means of X-ray crystallography [16,17]. Ecdysone receptors are limited to invertebrates that molt, which makes them well suited to regulating the expression of transgenes in medicine and agriculture [18,19]. It also makes them an attractive target for insecticide development. Indeed, bisacylhydrazine insecticides [20] exert their activity by binding to, and inappropriately activating, the ecdysone receptor, and these compounds exhibit remarkable selectivity across taxonomic orders [21,22]. Understanding fully the molecular basis of this speciWcity is a tantalizing goal, and may in turn lead to the development of novel ligand chemistries and insecticides with diVerent spectra of activity. In this article, we describe the puriWcation and properties of recombinant LBDs from the ecdysone receptors of four insects selected for their economic signiWcance as agricultural pests. The species span three taxonomic orders: Diptera (sheep blowXy, Lucilia cuprina), Hemiptera (peach aphid, Myzus persicae, and sweet potato whiteXy, Bemisia tabaci) and Lepidoptera (cotton bollworm, Helicoverpa armigera). In general, we chose to retain much or all of the D-segments in the recombinant proteins because of claims that the hinge regions of some nuclear hormone receptors – including ecdysone receptors – were required for LBD heterodimerization and/or ligand binding [23–26], and because recombinant LBDs bearing some or all of the cognate D-regions had given diVracting crystals [27,28]. The amino acid sequences of the expressed EcR DE/F segments share 81%, 57%, 60% and 61% identity, respectively, with the corresponding segment of D. melanogaster EcR (Supplementary Material, Section C). For each of the four species we co-expressed the recombinant LBD proteins in baculovirus-infected insect cells and puriWed the resulting LBD heterodimers in the absence of ligand. For three of the species, we also puriWed and characterized the corresponding ligand–LBD heterodimer complexes. The former preparations are useful for activity (i.e., ligand binding) assays that can be used to screen libraries of synthetic organic compounds, whereas the latter are suitable for structural determinations (e.g., X-ray crystallography). Both approaches can be used to guide the discovery and

design of selective ecdysone receptor agonists and antagonists, which in turn may form the basis of new and environmentally benign insecticides. Materials and methods Cloning and expression cDNAs encoding the EcR and USP proteins of the target insects were identiWed by screening high-quality cDNA libraries in Lambda ZapII (Stratagene) using homologous C-domain probes that had been PCR-ampliWed from the corresponding genomic DNA. Full-length sequences were cloned for L. cuprina [29,30], M. persicae, B. tabaci [17] and H. armigera. For each insect species the DE/F segments encoding the LBDs of EcR and USP were sub-cloned into pFastBac DUAL (Invitrogen). The recombinant EcR LBD was expressed using the polyhedrin promoter, and carried a hexahistidine tag at its N-terminus, while the recombinant USP LBD was expressed using the p10 promoter, and carried a FLAG tag at its N-terminus. In the case of the H. armigera receptor cDNA, PCR was used to prepare an additional construct that lacked the D-regions from both EcR and USP. Details for the preparation of the pFastBac DUAL constructs will be given elsewhere. Cassettes encoding paired EcR and USP segments were then transposed into bacmids for baculovirus construction and co-expression in Hi-5 insect cells; the boundaries of the expressed polypeptides are shown in Table 1. Large-scale recombinant protein production was typically achieved by infecting 4-5 L cultures of insect cells at a multiplicity of infection of 1–5 in a Celligen (New Brunswick ScientiWc) or Braun 6 L stirred bioreactor and culturing under controlled conditions (27 °C, 15–35 rpm) for 38–103 h. The recombinant insect cells were pelleted by centrifugation at 700g for 5 min, snap-frozen in liquid nitrogen, and stored at ¡70 °C. BuVers EcR40 buVer contained 25 mM Hepes, 40 mM KCl, 10% glycerol, 1 mM sodium EDTA, and 3 mM sodium azide, pH 7.0. NTA buVer contained 50 mM sodium phosphate, 10% glycerol, 0.3 M NaCl, 10 mM of 2-mercaptoethanol, 3 mM sodium azide, pH 7.4. MTBS buVer contained 50 mM Tris, 230 mM NaCl, 10% glycerol, and 3 mM sodium azide, pH 7.5. Extraction Manipulations were performed at 4 °C. For a typical ligand-free preparative extraction, 30–60 g of recombinant insect cells were suspended in EcR40 buVer and supplemented to provide 190 mL suspension containing 75 mM Hepes, 5.5 M leupeptin, 1.7 M pepstatin, 0.8 mM phenylmethanesulfonyl Xuoride (PMSF), 17 mM Na2S2O5, and 8 mM 2-mercaptoethanol, pH 7.0. Aliquots (45 mL) were sonicated 13 £ 5 s using an MSE 11 74.MK2 sonicator

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311

Table 1 Domain boundaries for recombinant polypeptides Species

Subunit

Segment

Amino acid boundaries

Cys

GenBank Accession No.

GenBank version

Nucleotides

Extra N-terminal sequencea

L. cuprina

EcR USP

DEF DE

Met-366 to Ala-757 Val-160 to Cys-467

10 9

U75355 AY007213

2 1

1839–3014 481–1404

MRGSHHHHHHGIR MDYKDDDDK

M. persicae

EcR USPb

DE DE/F

Pro-292 to Pro-599 Met-111 to Thr-377

5 4

EF174334 EF174335

1 1

1–924 1–801

MGHHHHHHR MDYKDDDDKP

B. tabaci

EcR USP

DE DE

Pro-120 to Ser-416 Lys-246 to Ser-496

7 5

EF174329 EF174330

1 1

1–891 1–753

MGMRGSHHHHHHR MDYKDDDDKGP

H. armigera

EcR USPc EcRd USPc

DE/F DE/F E/F E/F

Arg-252 to Leu-589 Lys-179 to Met-464 Leu-334 to Leu-589 Ser-202 to Met-466

9 4 5 4

EF174331 EF174333 EF174331 EF174332

1 1 1 1

1–1014 1–858 247–1014 70–864

MGMRGSHHHHHHRI MDYKDDDDKGP MGMRGSHHHHHHR MDYKDDDDKGP

We refer to the over-expressed polypeptides as recombinant LBDs, even though in most cases some or all of the hinge regions (D-segments) were also present. The terms DE/F and E/F denote individual recombinant LBDs where the presence of an F-region is uncertain; in the text, DE/F is also used in a collective sense to denote D-containing recombinant subunits (i.e., DEF, DE/F and DE polypeptides) as distinct from recombinant subunits without Dregions. Residue numbering comes from the full-length protein; in each case the C-terminal boundary is the natural C-terminus of the subunit. The number of Cys residues in each recombinant protein is shown in column 5. The amino acid sequences of the recombinant subunits are given in Supplementary Material, Sections A and B. a Extensions, shown using single-letter amino acid code, provided either a His6-tag tag (for recombinant EcR subunits) or a FLAG tag (for recombinant USP subunits) at the N-termini. b MpUSP1 isoform. c For technical reasons, the HaUSP2 isoform was used to construct the DE/F recombinant subunit and the HaUSP1 isoform was used to construct the E/F subunit. HaUSP1 contains 2 extra residues (not found in HaUSP2) in the region before the start of LBD helix 1. The two isoforms are believed to occur naturally, and the insertion is not expected to have any consequence for the function of the recombinant subunits (see Supplementary Material, Section B). d This recombinant subunit contained a S ! L substitution near the N-terminus (not recorded in the GenBank entry). Since it was located at a non-conserved solvent-exposed position remote from the ligand-binding pocket and heterodimerization interface, we did not consider it necessary to repair it (see Supplementary Material, Section A).

equipped with a 19 mm diameter probe. After adjusting to 0.4 M KCl, the lysate was ultracentrifuged at 100,000g for 1 h. The supernatant was dialyzed (Spectra/Por 1, Spectrum) for 3 h against 1.1 L EcR40 buVer containing 10 mM of 2-mercaptoethanol and clariWed by centrifuging at 14,000g for 30 min. It was then snap-frozen in liquid nitrogen and stored at ¡70 °C. If the intention was to prepare ligand–LBD heterodimer complex, then ponasterone A was present at 32 M in the lysis suspension and at 0.1 M in the dialysis buVer. Small extractions were similar but reduced in scale. For example, analytical extractions to compare (by ligand binding assay) the success of diVerent fermentations involved suspending 0.3 g of each cell pellet to provide 3 mL suspension containing protease inhibitors and 2-mercaptoethanol (without ligand) and sonicating for 15 £ 1 s using a 3 mm diameter tip. After adjusting to 0.4 M KCl, the lysate was centrifuged at 80,000g for 50 min. The supernatant was dialyzed (without ligand) as for preparative extractions but using a cellulose nitrate ultraWltration thimble (‘Collodion Bag’, Sartorius, Germany), and the supernatant was clariWed by centrifuging at 18,000g for 10 min. Standard puriWcations Manipulations were performed at 4 °C unless otherwise stated. Typically, for the immobilized metal aYnity chromatography (IMAC) puriWcation of an unliganded LBD

heterodimer a preparative extract (see above) was dialyzed (Spectra/Por 1) for 2 £ 3 h against 1.1 L NTA buVer, centrifuged at 14,000g for 20 min, and the supernatant was adjusted to 20 mM imidazole, pH 7.4. The sample was rotated with 3–6 mL bed volume of Ni–NTA agarose beads (Qiagen) at 10 rpm for 3 h, then centrifuged at 10,000g for 20 min. The beads were transferred to a 10 or 20 mL minicolumn and washed with 20 volumes NTA buVer containing 20 mM imidazole, pH 7.4, then eluted with 2 £ 0.75 volumes NTA buVer containing 250 mM imidazole, pH 7.4. The combined eluate was snap-frozen in liquid nitrogen and stored at ¡70 °C. PuriWcation of ligand–LBD heterodimer complexes was done as for unliganded heterodimers (described above) except that the cell extract contained 0.1 M ponasterone A and this ligand was maintained at 0.1 M in dialysis buVers, 0.5 M in IMAC wash buVer, and 3 M in IMAC elution buVer. Following IMAC puriWcation, typically the protein was dialyzed (Spectra/Por 1) 2 £ 3 h against 500 mL MTBS containing 10 mM of 2-mercaptoethanol, 0.5 M ponasterone and 2 mM CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate); then dialyzed overnight against 100 mL MTBS containing 10 mM of 2-mercaptoethanol, 2 mM CHAPS and a 10-fold molar excess of ponasterone A; and Wnally dialyzed 2 £ 3 h against 1 L MTBS containing 2 mM dithiothreitol (DTT) and 0.5 M ponasterone A. After supplementing to 3 M ponasterone A, the sample was concentrated to »0.5 mL by ultraWltration

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(MicroSep-10, Pall). The retentate was reduced for 2 h with 2 mM DTT and then puriWed by Fast Protein Liquid Chromatography (FPLC) gel Wltration (see next section). Eluate fractions corresponding to the main A280 peak were pooled and concentrated by ultraWltration (NanoSep-10, Pall). The retentate was supplemented to 3 M ponasterone A, sterilized by spin-Wltration (0.22 m, Costar Spin-X) and stored at 4 °C under nitrogen. Chromatography Procedures for IMAC are described in the previous section, and typically (appropriately dialyzed) IMAC eluate was applied to the columns described in this section. In all cases the protein content of the eluate was monitored at 280 nm (5 mm path length). Preparative gel Wltration of ligand–LBD heterodimer complexes (kept below 20 mg protein per separation) was done at 21 °C on a Superdex200 column (HR10/30, Pharmacia), equilibrated at 0.5 mL/ min in MTBS containing 2 mM dithiothreitol and 1 M ponasterone A. For calibration, BioRad gel Wltration standards (1.35, 17, 44, 158, and 670 kDa) were separated on the column in MTBS. Ion-exchange FPLC was done at 21 °C using a Mono-Q column (HR5/5, Pharmacia), or two such columns connected in series. Columns were equilibrated in 50 mM Tris buVer, 10% glycerol, 1 M ponasterone A, 3 mM sodium azide, pH 7.4 or 8.2, and developed at 1 mL/ min over 90 min with a salt gradient (0–0.7 M NaCl). The L. cuprina ligand–LBD heterodimer complex was immunopuriWed at 4 °C via the FLAG-tag on the USP subunit using anti-FLAG M2-agarose (Sigma) in MTBS containing 3 M ponasterone A, with elution by 0.5 mM FLAG. Hydrophobic interaction chromatography (HIC) was performed at 21 °C by FPLC using a Phenyl Superose column (HR5/5, Pharmacia) equilibrated in 50 mM sodium phosphate, 0.5 M ammonium sulfate, 1 mM DTT, 1 M ponasterone A, and developed at 0.5 mL/min over 1 h (0.5–0 M ammonium sulfate). Chemical and enzymatic modiWcations If a ligand–LBD heterodimer complex preparation was to be modiWed during puriWcation by alkylation of its free thiol groups, then the reaction was performed on the IMAC eluate prior to FPLC chromatography. To do this the IMAC eluate was Wrst supplemented to 0.5 mM PMSF and 2 mM DTT, and accessible cyst(e)ine side chains in the protein were fully reduced by incubating the mixture for 4 h at 23 °C under nitrogen. Alkylation was achieved by adding iodoacetic acid (from a 200 mM stock solution prepared fresh in 200 mM sodium hydroxide) or iodoacetamide (from a 200 mM stock solution prepared fresh in 4.12 mM sodium hydroxide) to a Wnal concentration of 12 mM (gentle alkylation) or 24 mM (stringent alkylation), and incubating in darkness for 23 h at 23 °C under nitrogen. To quench the reaction, the buVer capacity was Wrst strengthened by adding 0.15 vol. 1 M Tris pH 7.5, 2-mercap-

toethanol was added to 60 mM, and the mixture was incubated in darkness for 1 h at 23 °C. The alkylated sample was then dialyzed against the appropriate buVer for FPLC chromatography and puriWed by gel Wltration, ion exchange, or both (see previous section). Dephosphorylation of selected recombinant LBD heterodimer preparations prior to isoelectric focusing (IEF) was attempted by incubating samples (puriWed by IMAC followed by gel Wltration) at 4.4 mg protein/mL with 0.44 U/ l calf alkaline phosphatase (Roche) for 3 h at 37 °C in 215 mM Tris, pH 8.5, containing 22% (v/v) glycerol, with 440 M Mg2+ and 44 M Zn2+ as activators. Electrophoresis, IEF, and blotting SDS–PAGE was done at 100 V in Tris buVer using a 4% (w/v) acrylamide stacking gel (pH 6.8) and a 12% (w/v) acrylamide resolving gel (pH 8.8). Unless otherwise stated, samples were boiled for 3 min in loading buVer containing 5% (v/v) 2-mercaptoethanol. Coomassie Blue was PhastGel Blue R (Amersham). Gels were electroblotted onto nitrocellulose membrane (Protran 0.2 m, Schleicher & Schuell). The His6 peptide tag was detected using anti-polyhistidine monoclonal antibody (Sigma), while the FLAG tag was detected using anti-FLAG M2 monoclonal antibody (Sigma) or FLYTAG (AMRAD Biotech), all used at 1/ 1500 dilution. Antibody visualization was via horseradish peroxidase, using either the Amersham ECL or Roche BM chemiluminescence kits with Kodak Biomax MR Wlm. Densitometry of gels and autoradiograms was done using Kodak Digital Science 1D software, with background correction. Gels and blots were size calibrated using BioRad Pre-stained SDS–PAGE standards, whose actual molecular masses were lot-speciWc. Native PAGE was performed using the same buVer system as SDS–PAGE, but without SDS present and with an 8.5% (w/v) acrylamide resolving gel. Samples contained no SDS, were not boiled, and—unless otherwise stated—contained no reducing agent. For electrophoretic analysis of [3H]ponasterone A–LBD heterodimer complexes, native gel lanes were cut into transverse slices (8 £ 2 £ 1 mm) which were largely solubilized by overnight incubation at 37 °C in 0.3mL 100 vol. H2O2 solution. To complete the process, a further 0.1 mL H2O2 was added and the incubation continued for another 5 h, whereupon addition of 7 mL Instagel Plus (Packard) enabled the dissolved slices to be scintillation counted for tritium (1 min/ sample, Packard TriCarb 2100TR). IEF gels were run in a Model 111 Mini IEF Cell (BioRad). Gels were hand-poured using 24.25% (w/v) acrylamide, 0.75% (w/v) bis-(N,N⬘-methylene-bis-acrylamide) (T D 25%, C D 3%), and Bio-Lyte 3/10 ampholytes (BioRad). Unalkylated samples contained DTT as reducing agent. Samples were focused in the absence of denaturants for 15 min at 100 V, followed by 15 min at 200 V and 60 min at 450 V. Focused gels were Wxed and stained using a mixture of CuSO4, Crocein Scarlet and Coomassie Brilliant Blue R-250 (BioRad).

L.D. Graham et al. / Protein Expression and PuriWcation 53 (2007) 309–324

Assays Protein concentrations were measured by Pierce Coomassie Plus assays done in 96-well plates and calibrated using bovine serum albumin (BSA), which gave results within 6% of those determined by quantitative amino acid analysis (data not shown). Molar concentrations of LBD were calculated using the sum of the expected molecular masses for the constituent EcR and USP polypeptides (Table 3). The solvent-accessible thiol content of a protein preparation was assessed by mixing a 20 l sample with 0.5 mL of ice-cold 1 mM 5,5⬘-dithiobis(2-nitrobenzoic acid) in 0.5 M Tris, pH 8.0. The absorbance of the resulting solution was immediately measured at 412 nm and the thiol concentration calculated using  D 12,800 M¡1cm¡1 for the yellow leaving group, the 2-nitro-5-mercaptobenzoate dianion [31]. The ligand binding assay, which used [3H]ponasterone A ([24,25,26,27-3H(N)]ponasterone A, 150–200 Ci/mmol, NEN Life Science Products) as ligand, was adapted from Koelle et al. [6]. The standard assay involved incubating 2.2 nM bindable [3H]ponasterone A (i.e., 2.2 nM ligand after correcting for the fraction of radioactive material that could not be bound even in the presence of excess LBD heterodimer) with recombinant LBD heterodimer in the presence of BSA (0.5 mg/mL) for 90 min at 21 °C. Recombinant protein, including any [3H]ponasterone A–LBD heterodimer complex present, was captured on GF/C glass Wber discs (Whatman) and unbound ligand was removed by washing under suction with ice-cold EcR40 buVer. Airdried Wlters were scintillation counted for tritium in 7 mL InstaGel Plus (Packard TriCarb). The assay, and associated equilibrium binding experiments, will be described in greater detail elsewhere (Graham et al., in preparation). Assays that test for the eVect of CHAPS are described in Supplementary Material, Section D. Mass spectrometry Samples were IMAC-puriWed recombinant LBD heterodimer preparations at 1.5–11.3 mg protein/mL in 25 mM ammonium bicarbonate solution containing 0.5 mM DTT. Matrix-assisted laser desorption ionization time-of-Xight (MALDI-TOF) mass spectra were acquired in linear mode using a Bruker ReXex MALDI-TOF-MS Wtted with a delayed extraction ion-source and a high mass detector. Samples were co-crystallized with sinnapinic acid on a Scout 26 target and irradiated with a nitrogen laser; ions were accelerated at 20 kV following a 20 s delay. Spectra were calibrated externally using the Xight times of BSA ions carrying 1, 2 and 3 charges. Electrospray (ESI) mass spectra were recorded in positive ion mode using a VG Platform Mass Spectrometer equipped with a Jasco pump and autosampler. Samples were applied at 40 l/min with pure water as running solution. The instrument supplied a cone voltage ramp of 50–100 V over the m/z range 500–2000 Da, and a capillary voltage of 3.2 kV. The Maxent algorithm was used

313

to deconvolute spectra for ions with multiple charges and arrive at true molecular mass values. MALDI-TOF methods for identifying host cell proteins from the masses and fragmentation patterns of their tryptic peptides are described in Supplementary Material, Section F. Results Expression and activity For each of the four insect species, the DE/F segments of their EcR and USP subunits were co-expressed as aYnitytagged proteins using a baculovirus expression construct (Table 1). A construct co-expressing the tagged E/F segments of the H. armigera receptor subunits was also prepared (Table 1). For convenience we will refer to all of the baculovirus-expressed polypeptides as recombinant ligand binding domains (LBDs), even though the DE/F versions also contain most or all of the hinge regions (D-segments). Large-scale (4–5 L) fermentations of recombinant insect cells yielded 30–100 g wet cells, which—assuming 60% yield and 80% purity for the immobilized metal aYnity chromatography (IMAC) step, see below—typically contained 0.3– 1.6 mg of recombinant protein per gram of cells. Yields of IMAC-puriWed protein (puriWed with or without ligand) ranged 2.9–16.3 mg/L culture over 16 puriWcations from 10 fermentations, with a median value of 7.6 mg/L culture. All of the recombinant heterodimers were active in the ligand binding assay; the highest speciWc activities for [3H]ponasterone A binding by DE/F- and E/F-containing cell extracts ranged 20–360 pmol/mg protein. Recombinant cell extracts containing the L. cuprina LBD heterodimer typically had a speciWc [3H]ponasterone A binding activity of »85 pmol/ mg protein, approximately 10,000 times higher than that of L. cuprina embryo extracts (0.009 pmol/mg protein) whose ecdysteroid-binding capability reXected high natural levels of the full-length receptor. Standard binding assays done on recombinant cell extracts indicated that the highest expression obtained during the project was equivalent to 16.6 mg active LBD heterodimer per liter of culture, a value obtained with the L. cuprina protein. Most of the baculovirus constructs produced approximately equal amounts of recombinant EcR and USP, although for the M. persicae proteins Western blots indicated a signiWcant excess of recombinant USP over EcR (see below). PuriWcation and stability The recombinant LBDs were equipped with N-terminal aYnity tags to facilitate their detection and puriWcation. Even in the absence of ligand, all Wve heterodimers could be aYnity-puriWed from cell extracts using a nickel chelate resin (IMAC) to capture the His6-tag of the recombinant EcR LBD. Data for IMAC puriWcations of various apoLBD (i.e., unliganded LBD) heterodimers are reported individually in Table 2a; densitometry of Coomassie-stained bands conWrmed that the IMAC eluates contained an

314

L.D. Graham et al. / Protein Expression and PuriWcation 53 (2007) 309–324

Table 2 PuriWcation tables for recombinant LBD heterodimers SpeciWc activityb (pmol/g)

Enrichment (-fold)

Yieldc(%)

Purityf(%)

(a) IMAC puriWcations of ligand-free LBD heterodimers L. cuprina, 1.6 g cells Extract 72 5.059 IMAC 0.79 0.801

0.070 1.014

— 14.5

[100] 16d,e

1.5g 81

L. cuprina, 8.8 g cells Extract 429 IMAC 5.2

39.1 8.43

0.091 1.620

— 17.8

[100] 22d,e

1.8g 89

M. persicae, 37 g cells Extract 1665 IMAC 17

82.9 21.1

0.050 1.239

— 24.8

[100] 25

1.4g 80

B. tabaci, 42 g cells Extract 2024 IMAC 25.2

62.3 18.8

0.031 0.744

— 24.0

[100] 30

2.1g 95

Protein (total, mg)

Activitya (total, nmol)

Protein (total, mg) (b) Multi-step puriWcation of a ponasterone A–LBD heterodimer complex Extract 2688 IMAC (Ni–NTA agarose) 22.6 13.5 Alkylation (iodoacetamide)i 9.3 Ion exchange (Mono-Q)j Gel Wltration (Superdex-200)i 6.8

Purityf (%)

Yieldh (%)

1.0g 70 — 86 97

— [100] 60 51 42

In (a), the starting mass of wet recombinant insect cells is shown after the species name. Since these were standard IMAC puriWcations of apo-LBD heterodimer, no ligand or carrier protein was present. All values in (a) are for ligand binding by cell extracts and IMAC eluates containing DE/F heterodimers in the absence of CHAPS. Square brackets denote assigned values. In (b), the data are for 56 g of wet cells containing M. persicae recombinant heterodimer, which was puriWed and alkylated in the presence of saturating concentrations of ponasterone A. Square brackets denote assigned values. a Binding capacity for [3H]ponasterone A, measured by standard ligand binding assay. b pmol [3H]ponasterone A bound per g protein, measured by standard ligand binding assay. c Based on activity, measured by standard ligand binding assay. d In contrast to the activity yields, densitometry of immunoblot autoradiograms indicated that yields of recombinant protein were 67–88%, and estimates based on Coomassie-stained band intensities were 50–60%. e The proportion of heterodimer that failed to bind to the IMAC resin was 5–22% by activity assays and »17% by densitometry of immunoblot autoradiograms. f Based on densitometry of reduced samples resolved on Coomassie-stained SDS–PAGE gels. g Since the recombinant bands could not be resolved suYciently from host cell proteins to allow densitometry, this value was inferred on the basis that the IMAC step gave 60% yield of recombinant protein (see note d) at the % purity shown in the table. h Amount of heterodimer protein, calculated as (total protein £ % purity/100) and expressed as a percentage of the amount of heterodimer in the IMAC eluate (calculated in the same way). i Alkylation yield includes sample reduction, reaction quench, and several dialysis and centrifugation steps; gel Wltration yield includes dialysis, centrifugation, concentration and spin-Wltration. j The elution proWle for this particular sample is shown in Fig. 1a.

equimolar ratio of recombinant EcR and USP subunits. Further puriWcation could be achieved by subjecting IMAC-puriWed recombinant proteins to FPLC ionexchange chromatography or gel Wltration (Fig. 1). For example, in the pH range 7.5–8.2, M. persicae ligand–LBD heterodimer complexes eluted from MonoQ at 125– 200 mM NaCl (Fig. 1a). Both column chromatography steps were eYcient (>65% step yields for recombinant protein) and inexpensive. It was possible to use both in sequence (Table 2b), but the combination of IMAC followed by a single FPLC step was usually suYcient. ImmunopuriWcation by FLAG-tag capture was also possible, but oVered no advantages to oVset the high cost and fragility of the resin (data not shown). Hydrophobic interaction chromatography (HIC) of IMAC-puriWed L. cuprina LBD heterodimer–[3H]ponasterone A complex on a Phenyl

Superose HR5/5 column resulted in a release of free ligand and low (»20%) recovery of the recombinant protein, which now contained more USP than EcR subunits (data not shown). To obtain apo-LBD heterodimer preparations for ligand binding experiments, 1–42 g of recombinant cells were lysed by sonication and the recombinant heterodimer was puriWed from the clariWed lysate by IMAC in the absence of any ligand. In view of the eYcient capture (78–95%) of activity and recombinant protein by the IMAC matrix, the eluted activity yields (16–30%) were unexpectedly low, and considerably lower than the yields of recombinant protein (50– 88%) estimated from gel and immunoblot band intensities (Table 2a). Moreover, direct comparisons of small-scale IMAC puriWcations showed that the activity yields of L. cuprina apo-LBD heterodimer increased from 37% to 64%, with-

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315

Fig. 1. FPLC chromatography of IMAC-puriWed ligand–LBD heterodimer complexes. (a) Ion exchange. In this example, 13.5 mg iodoacetamide-modiWed M. persicae LBD heterodimer–ligand complex was separated by Mono-Q (2 £ 1 mL columns in series) at pH 8.2. The A280 trace (left-hand scale) monitors the elution of protein while the straight line shows the applied NaCl concentration (right-hand axis); eluate volume includes the wash step. The main peak (fractions B–D) contained 9.3 mg protein (69% step yield). Inset: SDS–PAGE of reduced samples: M, molecular mass marker proteins with actual sizes of 205, 113, 75, 54, 35, 29, 21, 7 kDa; L, load sample; A–D, eluate fractions. (b) Gel Wltration. Here 11.7 mg iodoacetamide-modiWed M. persicae LBD heterodimer–ligand complex was separated by Superdex-200 (bed volume 24 mL). The A280 trace (right-hand scale) monitors the elution of protein; INJ indicates the point at which the sample was injected onto the column. The main peak (A) contained 7 mg protein (60% step yield). Inset: SDS–PAGE of reduced samples: M, molecular mass marker proteins; A, peak. Load sample is as for (a).

out any change in the yield of recombinant protein, when a carrier protein (0.5 mg/mL BSA) was included in the wash and elution buVers. In combination, these results suggest that ligand-free LBD heterodimers are fragile and easily become inactivated during preparative manipulations. Using the standard [3H]ponasterone A binding assay, the speciWc activity values for L. cuprina, M. persicae and B. tabaci apo-LBD heterodimers prepared by standard IMAC puriWcations (including those shown in Table 2a) were 1.0–1.6, 1.1–1.3 and 0.74 pmol/g protein, respectively. Scintillation counting showed that, after washing, the GF/C discs used in the binding assay retained a similar amount of [3H]ponasterone A– LBD heterodimer complex to washed Ni–NTA-agarose beads (data not shown; also Supplementary Material, Section D), with losses estimated at up to »30%. After correcting for this, the mean speciWc activity values suggested that only 7–18% of the heterodimers in each preparation were functional (Table 4). We noticed that dilute preparations of puriWed apo-LBD heterodimers were particularly labile, in that the recombinant proteins denatured and precipitated during a single freeze-thaw cycle. Immunoblots and activity assays (not shown) conWrmed that a carrier protein (0.5 mg/ mL BSA) fully protected dilute apo-LBD heterodimer solutions against loss during freeze-thaw. Saturating the LBD heterodimers with ligand also rendered them more stable. In the absence of a carrier protein, immunoblots (not shown) revealed that little or no puriWed L. cuprina apo-LBD heterodimer remained in solution when a dilute sample (0.12 mg/ mL) was stored at ¡70 °C for 48 days, whereas >70%

remained soluble in the presence of 30 M ponasterone A. Ponasterone A also appeared to improve the survival of functional LBD heterodimers during puriWcation. For example, when a recombinant cell extract containing L. cuprina LBD heterodimer was saturated with [3H]ponasterone A (65 Ci/mol) and the IMAC-captured ligand–heterodimer complex was washed in the presence of the same ligand (100 nM), scintillation counting of the IMAC eluate indicated a [3H]ponasterone A content equivalent to 71% of the LBD heterodimer population being active (Table 4). In contrast, when L. cuprina apo-LBD heterodimers were puriWed by IMAC in the absence of ponasterone A, activity assays suggested that on average only 18% of the recombinant molecules were functional (Table 4). To prepare ligand–LBD heterodimer complexes for crystallization trials, 60–70 g batches of recombinant cells were sonicated in the presence of excess ponasterone A and the recombinant complex was puriWed from the clariWed lysate using IMAC followed by an FPLC chromatography step—usually gel Wltration (Fig. 1b)—in the presence of saturating concentrations of ponasterone A. In many puriWcations the LBD heterodimer was exposed transiently to 2 mM CHAPS in order to enhance ligand binding (see below) and encourage the removal of contaminating proteins, but CHAPS was never present in the Wnal eluate as it was thought that this might hinder crystallization. Whenever a ligand–LBD heterodimer complex was puriWed for crystallization trials, a small amount of the relevant batch of recombinant cells was extracted separately (in the

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Table 3 Molecular mass and pI data for recombinant proteins Species

Subunit

Recombinant segment

Expected M ra

Apparent Mra (SDS–PAGE)b

Observed Mra (mass spec)c

Calculated pId

L. cuprina

EcR USP

DEF DE

45.5 35.4

55 39

45.7 35.6

M. persicae

EcR USP

DE DE/F

36.7 31.5

42 34

36.6 31.5

B. tabaci

EcR USP

DE DE

35.8 30.0

42, 40 (43, 38) 38 (37)

36.0g 30.1g

H. armigera

EcR USP EcR USP

DE/F DE/F E/F E/F

40.5 33.7 30.8 31.3

50 36 (35) 35 34 (44)

n.d.h n.d. n.d. n.d.

} 6.0 5.8 } 6.5 5.1 } 6.7 5.6 } 6.3 5.2 } 6.3 6.8

Heterodimer, observed pI (native IEF)e

Heterodimer, observed Mra (gel Wltration)f

6.3–6.8 (4–5)

70–130 (92)

6.2–6.5 (5–6)

90–110

6.3–6.9 (5–7)

85–100

6.4–7.1 (7)

n.d.

6.6–6.9 (3)

n.d.

a Size values are in kDa. Expected values were calculated for recombinant polypeptides with aYnity tags; likewise, the observed data are mean values for tagged recombinant proteins. b Apparent molecular mass values are calculated from gels of reduced samples developed by Coomassie staining or immunoblot (or, in parentheses, from autoradiography of reduced in vitro transcription/translation products obtained from a plasmid expressing the tagged subunit on its own). c Values are m/z values obtained by MALDI-TOF or, for values in italics, mass values obtained by ESI. d Calculated using Pepstats, with aYnity tags and terminal charges included. e Observed pI values are for the main group of bands seen in non-denaturing IEF of the heterodimer in the presence of ponasterone A (or, for the H. armigera LBD heterodimers, in its absence); an estimate of the number of discrete isoforms in this group is given in parentheses. f Non-denaturing gel Wltration of receptor–ponasterone A complexes was monitored either by absorbance at 280 nm for IMAC-puriWed ponasterone A–LBD heterodimer complexes (or, in parentheses, by elution of radioactivity for a [3H]ponasterone A–LBD heterodimer complex). g The spectrum also had strong peaks at 25.8 and 23.6 kDa, which did not correspond to any bands seen in subsequent SDS–PAGE of the sample. h Not determined.

absence of ligand and CHAPS) and subjected without further puriWcation to ligand binding assays to determine the binding activity per gram of cells. From this and the mass of cells taken, we could infer the ligand binding capacity present at the start of each large-scale puriWcation of ligand–LBD heterodimer complex. When this value was adjusted for likely losses (assuming that the LBD heterodimers were now stable because they had bound ligand) and correlated with the amount of protein obtained from the preparative IMAC step, the recombinant L. cuprina, M. persicae and B. tabaci complexes gave mean values of 1.6 § 0.1 (n D 2), 4.1 § 0.8 (n D 6) and 1.5 § 0.2 (n D 3) pmol [3H]ponasterone A per g protein, respectively. Treated as before, these data equate to mean values of 17–50% for the proportion of functional molecules in preparations of ligand–LBD heterodimer complexes (Table 4). From the foregoing, it is clear that there was scope for improvement in the ligand binding stoichiometry of puriWed apo-LBD heterodimers. To our surprise, when the zwitterionic detergent CHAPS was included at 2 mM in standard ligand binding assays, it typically increased several-fold the amount of [3H]ponasterone A bound by puriWed recombinant LBD heterodimer preparations (Supplementary Material, Section D). The same eVect was observed when ligand–LBD heterodimer complexes were captured using Ni–NTA-agarose beads rather than glass Wber discs (Supplementary Material, Section D). In equilibrium binding studies done in the absence and presence of 2 mM CHAPS, the Kd value for a puriWed recombinant M. persicae LBD heterodimer preparation remained unchanged (Graham et al, in preparation), whereas in the

presence of CHAPS the Rt value (i.e., the number of functional ligand binding sites) per unit volume increased from 245 § 10 pM to 322 § 29 pM. Purity and integrity Mass spectra for puriWed recombinant L. cuprina and M. persicae LBD preparations are shown elsewhere (Supplementary Material, Section E). Expected and observed molecular mass values for each recombinant LBD polypeptide are compared in Table 3. In mass spectrometry the observed sizes for EcR were typically slightly smaller than expected, while those for USP were slightly larger, and the ion signal intensities for EcR were lower than those for USP. In SDS–PAGE, the recombinant LBDs typically ran slightly slower than expected. Fig. 2a shows representative Coomassie-stained gel lanes for the recombinant proteins from each insect species, and Fig. 2b illustrates the immunoblot results. The expected and observed molecular mass values for each polypeptide are compared in Table 3. For all species, SDS–PAGE showed that a small amount of a 75 kDa contaminant co-puriWed with the recombinant LBD heterodimers; this was true for IMAC, Mono-Q and Superdex-200 chromatography (Figs. 1 and 2a). From the masses and fragmentation patterns of its tryptic peptides, this protein was identiWed as Hsc70 from the Hi-5 cells used as expression host (Supplementary Material, Section F). In addition, puriWed samples of the B. tabaci LBD heterodimer contained a signiWcant contaminant at 44 kDa (Fig. 2a, lane 3). Unexpectedly, IMAC-puriWed preparations of H. armigera E/F recombinant protein (Fig. 2a, lane 5)

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317

Fig. 2. SDS–PAGE of puriWed recombinant LBD heterodimers. All samples contained reducing agent. Letters indicate intact recombinant EcR (E) and USP (U) subunits, nicked versions of recombinant EcR (A) and USP (B), and the 75 kDa (C), 44 kDa (D) and 57 kDa (F) contaminants mentioned in the text. Bands E and A are those seen in immunoblots using anti-His6 antibody alone, bands U and B are those seen in blots using anti-FLAG antibody alone, while bands C–E were not seen in immunoblots. M, marker proteins, showing average molecular masses in kDa (see Materials and methods for details). (a) Coomassie-stained gels. Preparations from IMAC followed by gel Wltration (lanes 1–3) or from IMAC alone (lanes 4–5). L. cuprina heterodimer (lane 1). M. persicae heterodimer (lane 2). B. tabaci heterodimer (lane 3); apparent molecular masses for the weaker and stronger bands at E are 42 kDa and 40 kDa, respectively. H. armigera DE/F heterodimer (lane 4). H. armigera E/F heterodimer (lane 5), showing some aggregated protein that failed to enter the resolving gel. (b) Immunoblots. Preparations were IMAC eluates (lanes 1, 3–5) or cell extracts (lane 2). Unless stated otherwise, blots were developed using a mixture of anti-His tag and anti-FLAG antibodies. L. cuprina heterodimer (lane 1). M. persicae heterodimer (lane 2); note the excess of recombinant USP over EcR subunits. B. tabaci heterodimer, anti-His6 antibody only (lane 3); the upper and lower band of the doublet (whose signals have largely fused) correspond to the upper and lower bands at E in (a), lane 3. H. armigera DE/F heterodimer (lane 4). H. armigera E/F heterodimer (lane 5). The hollow centers to the H. armigera bands (lanes 4 and 5) are caused by exhaustion of the chemiluminescence substrate in the areas of greatest signal intensity.

contained relatively large amounts of a 57 kDa contaminant that was present only in trace quantities in preparations of the H. armigera DE/F recombinant. From the masses and fragmentation patterns of its tryptic peptides, the contaminant was identiWed as an -tubulin (Supplementary Material, Section G). Another diVerence between IMAC-puriWed H. armigera DE/F and E/F preparations was that the latter contained some high molecular mass material that failed to enter the resolving (and sometimes even the stacking) gel in SDS–PAGE of in reduced samples. It appeared to consist of non-disulWde-mediated protein aggregates. Immunoblots, which only addressed bands within the resolving gel, provided no indication of whether or not this material contained recombinant polypeptides. Despite the presence of protease inhibitors in the lysis buVer, Coomassie-stained gels revealed that a small proportion of the recombinant subunits of the L. cuprina and M. persicae heterodimers usually underwent proteolytic nicking. Chemical modiWcation of the samples (described below) or the inclusion of protease inhibitors after the aYnity capture step did not prevent this (data not shown). In preparations that had been polished by FPLC chromatography, degradation would continue slowly upon storage at or above 4 °C. With L. cuprina preparations, recombinant EcR was degraded about twice as rapidly as recombinant USP, yielding a stable fragment that migrated with an apparent molecular mass of 42 kDa (Fig. 2, band A) and was visible in immunoblots targeting the N-terminal His6tag (Fig. 2b). With M. persicae preparations, recombinant EcR was largely resistant to proteolysis but the recombinant USP subunit succumbed at a rate comparable to recombinant L. cuprina USP. Recombinant L. cuprina and M. persicae USP each yielded a stable proteolysis fragment that was signiWcantly smaller than the original (Fig. 2a, bands B); immunoblots showed that the N-terminal Flag

tag was still present in both (Fig. 2b, bands B). Since the long fragments of the recombinant EcR and USP subunits co-puriWed with intact polypeptides in various non-denaturing chromatography steps (Figs. 1 and 2), and since other experiments (not shown) demonstrated that the column was well able to resolve free recombinant LBD monomers from recombinant LBD heterodimers, it seemed that C-terminal nicking did not compromise heterodimer assembly. The fact that the recombinant B. tabaci LBDs always appeared as a triplet in SDS–PAGE (Fig. 2a, lane 3) suggested that this heterodimer too might be susceptible to protease activity. PuriWed preparations of this protein underwent substantial proteolysis during prolonged crystallization trials of the ponasterone A–LBD heterodimer complex; Coomassie-stained gels of crystals obtained after 3 months at room temperature only showed bands at 30, 27, 24 and 23 kDa (not shown). DisulWde bond formation Each of the recombinant subunits contains a number of Cys residues (Table 1). In non-reducing conditions, we found that disulWde bonds could form both within an LBD (constraining the polypeptide so that it migrated slightly faster in SDS–PAGE) and between LBDs (creating subunit oligomers which in some cases could barely enter the resolving gel). Fig. 3 shows SDS–PAGE of air-oxidized recombinant samples run in the absence and presence of reducing agent. The origins of the various disulWde-containing species could readily be assigned on the basis of changes in band intensities and mobilities, and these identiWcations were subsequently conWrmed by immunoblotting (e.g., Fig. 3c). Intramolecular disulWde bonds were observed for recombinant EcR subunits (L. cuprina and M. persicae) and USP subunits (some, but not all, preparations of

318

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Fig. 3. DisulWde bond formation within and between recombinant LBDs. Gels are SDS–PAGE. Letters indicate marker proteins (M) with molecular masses shown in kDa, recombinant EcR subunits without disulWde bonds (E), and recombinant USP subunits without disulWde bonds (U). Symbols indicate recombinant USP with one or more intramolecular disulWde bonds (»), recombinant EcR with one or more intramolecular disulWde bonds (–), disulWde-bonded recombinant EcR dimers (¤), and disulWde-bonded recombinant EcR multimers near the top of the resolving gel (冷). (a) Coomassie-stained gel of L. cuprina complex. Ponasterone A–LBD heterodimer complex was puriWed by IMAC (lanes 1 and 3) or IMAC followed by M2-agarose (lane 2), and the preparations (0.6–2.7 mg protein/mL) were allowed to air-oxidize by storing with little or no reducing agent. Samples for lanes 1 and 2 were not reduced before loading. Mild oxidation (2 months, ¡70 °C) allowed intramolecular disulWde bonds to form in both recombinant subunits (lane 1), whereas more severe oxidation (3 months, 4 °C) resulted in multimerization of most of the recombinant EcR subunit (lane 2). Boiling either sample in the presence of 2-mercaptoethanol before loading abolished both intramolecular and intermolecular bonds (lane 3). (b) Coomassie-stained gel of M. persicae complex. Ponasterone A–LBD heterodimer complex was puriWed by IMAC (lane 2) or IMAC followed by gel Wltration (lanes 1 and 3), then allowed to air-oxidize by storing without reducing agent. Samples 1–3 were not reduced before loading. A sample that had been stringently alkylated with iodoacetamide and stored at high concentration (39 mg/mL, 21 days, 20 °C) showed almost no disulWde bond formation (lane 1). A less concentrated unalkylated sample (5.2 mg/mL, 6 days, 4 °C) showed extensive evidence of intramolecular disulWde bonding (lane 2). A highly concentrated unalkylated sample (24 mg/mL, 5 days, 4 °C) showed extensive evidence both of intramolecular bonding (as before) and intermolecular bonding, with particularly prominent recombinant EcR dimer and multimer bands (lane 3). Boiling either of the last two samples in the presence of 2-mercaptoethanol before loading abolished both intramolecular and intermolecular bonds (lane 4). (c) Immunoblot of M. persicae complex. Sample similar to (b) lane 2 after Coomassie staining (lane 1) or development of an immunoblot with antibody speciWc for the His6-tag on the recombinant EcR subunit (lane 2).

L. cuprina; compare Fig. 3a, lanes 1 and 2). In contrast, intermolecular bonds were observed only for recombinant EcR subunits (all four insect species). Somewhat surprisingly, multimer formation in H. armigera E/F samples involved the complete recruitment of the prominent 57 kDa contaminant into the high molecular mass material (not shown). DisulWde bond formation did not seem to impact greatly on ligand binding: for instance, a sample of IMACpuriWed M. persicae protein that had been extensively airoxidized (so that 745% and »25% of its recombinant EcR was involved in intramolecular and intermolecular disulWde bonds, respectively) showed only »18% decrease in ligand binding activity relative to a fully reduced control sample. DisulWde-mediated oligomerization could be suppressed by using thiol-speciWc reagents (iodoacetic acid or iodoacetamide) to alkylate accessible Cys residues. In pilot experiments, gentle S-carboxymethylation of L. cuprina LBD heterodimer saturated with [3H]ponasterone A (65 Ci/mol) underwent a 38% decrease in accessible thiol content with only a slight (13%) loss of bound ligand (data not shown). Iodoacetamide-modiWed recombinant proteins were less soluble than their unalkylated or S-carboxymethylated counterparts, especially below pH 7.4; their propensity to precipitate underlies the low step yield for alkylation in Table 2b. Gentle alkylation was sometimes, but not always, suYcient to prevent subsequent oligomerization of the recombinant EcR subunits, whereas stringent alkylation invariably prevented oligomer formation (e.g., Fig. 3b, lane 1). Alkylation could

cause a slight decrease in recombinant polypeptide mobility in SDS–PAGE (not shown); the eVect was more noticeable with iodoacetic acid, the EcR subunit, and unreduced samples. Although it was not evident from SDS–PAGE, mass spectrometry revealed that modiWcation with either reagent introduced substantial mass heterogeneity into the recombinant proteins (Supplementary Material, Section E). Non-denaturing gels Native PAGE performed in high-pH discontinuous gels with puriWed L. cuprina [3H]ponasterone A–LBD heterodimer complex gave a series of bands with the appearance of an oligomer ladder (Fig. 4a). In keeping with this interpretation, immunoblots conWrmed that each of the bands had a similar—approximately equimolar—recombinant EcR: USP subunit ratio (Fig. 4a). The main band, which had the highest mobility, was presumed to be the ‘monomer’, i.e. the ligand–EcR–USP complex. The presence of 2-mercaptoethanol or DTT greatly decreased the proportion of material in the slower-moving bands (e.g., Figs. 4b and 4c). Scintillation counting of dissolved gel sections showed that [3H]ponasterone A was associated speciWcally with the main LBD heterodimer band and also, if present, with the slower-moving bands (Fig. 4a). Coomassie-stained lanes containing the unliganded apo-LBD heterodimer (not shown) were indistinguishable from those containing the ligand–LBD heterodimer complex.

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319

Fig. 4. Non-denaturing PAGE of puriWed recombinant LBD heterodimers. The polarity of the gels is indicated to the left of the Wrst panel. (a) Ligand– LBD heterodimer complex remains assembled. ExempliWed here by IMAC-puriWed L. cuprina ponasterone A–LBD heterodimer complex, prepared without reducing agent. Immunoblots using antibodies against FLAG alone (lane 1) or His tag alone (lane 2) produce an identical pattern, namely a major band corresponding to the ponasterone A–LBD heterodimer complex (A) and decreasing amounts of less mobile species (B, C) that are likely to be dimers and trimers, respectively, of the complex. BSA (lane 3) and [3H]ponasterone A–LBD heterodimer complex (lane 4) visualized by Coomassie staining; the BSA (which ran as a ladder of monomer, dimer, trimer, etc.) provided a useful reference for comparing gels. A repeat of lane 4 was run immediately to its right; this was sliced into sections (nicks corresponding to the end of each slice boundary can be seen at the right of lane 4), and the amount of [3H]ponasterone A in each slice was determined by scintillation counting (bar graph). For each of the bands A–C there is a peak of radioactivity (marked) whose relative magnitude reXects the relative band intensity seen in gels and blots. (b) Alkylated ligand–LBD heterodimer complexes diVering in net charge. ExempliWed here by IMAC-puriWed L. cuprina ponasterone A–LBD heterodimer complex, modiWed with iodoacetamide (lane 1) or iodoacetic acid (lane 3). The unalkylated control sample (lane 2) had been taken through the modiWcation procedure but without adding iodoacetamide or iodoacetic acid; all three samples contained reducing agent from the quench step (see Materials and methods for details). (c) LBD heterodimers from diVerent species. A reference track of BSA (lane 1) is followed by (unalkylated and ligand-free) puriWed recombinant heterodimers from L. cuprina (lane 2) and M. persicae (lane 3). The LBD heterodimer samples were puriWed by IMAC (lane 2) or IMAC followed by gel Wltration (lane 3), and both contained reducing agent (17 mM DTT).

Fig. 5. Non-denaturing isoelectric focusing (IEF) of puriWed recombinant LBD heterodimers. Marker proteins (M) spanned the range pI 4.5–9.6, but since LBD heterodimer preparations contained no bands above pI 8.2, the top part of each gel has been omitted. (a) Unalkylated LBD heterodimers. For lanes 1–3 the recombinant heterodimers had been puriWed with ponasterone A present, whereas for lanes 4–5 the receptor had been puriWed in the absence of ligand. L. cuprina complex, puriWed by IMAC (lane 1). M. persicae complex, puriWed by IMAC followed by gel Wltration (lane 2). B. tabaci complex, puriWed by IMAC (lane 3). H. armigera DE/F heterodimer, puriWed by IMAC (lane 4). H. armigera E/F heterodimer, puriWed by IMAC (lane 5); the doublet near the top of the gel (pI »7.9) is likely to be the 57 kDa contaminant (band F, Fig. 2a). (b) EVect of alkylation. Both sample lanes show L. cuprina ponasterone A–LBD heterodimer complex, puriWed by IMAC followed by gel Wltration. Gentle alkylation with iodoacetic acid (lane 1) caused the bands to focus at a lower pI than the unalkylated complex (cf. (a), lane 1), whereas gentle alkylation with iodoacetamide (lane 2) did not.

Unlike in SDS–PAGE, protein mobility in native gels depends on charge as well as size. Thus, with the recombinant L. cuprina heterodimer, native PAGE was able to separate the iodoacetic acid-modiWed, iodoacetamide-modiWed, and unalkylated forms of the ponasterone A–LBD heterodimer complex from each other on the basis of their diVerent net charges (Fig. 4b). In a comparison of unalkylated LBD heterodimers from diVerent species, the recom-

binant M. persicae heterodimer migrated as a discrete band at 1.3 times the rate of the main L. cuprina band (Fig. 4c). Non-denaturing IEF gels revealed multiple isoforms for each LBD heterodimer (Fig. 5a). Table 3 shows the theoretical pI values for each recombinant subunit, and the pI ranges observed for the various recombinant heterodimers over several independent puriWcations. Alkaline phosphatase treatment of recombinant L. cuprina and M. persicae

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proteins had no eVect on their IEF patterns (not shown), suggesting that the isoforms were not the result of diVerential phosphorylation. As expected for a non-secreted protein, enzymatic treatments of a puriWed recombinant LBD heterodimer to remove N-glycans and sialic acid residues had no eVect on its IEF pattern (data for M. persicae; not shown). For each LBD heterodimer, alkylation with iodoacetic acid shifted the set of bands to a more negative region, whereas iodoacetamide had no eVect (Fig. 5b). Discussion Expression Recombinant nuclear hormone receptors and their LBDs have usually been expressed using Escherichia coli as host [32], but poor solubility of the LBDs has often required them to be modiWed [32]. However, precedents suggested that baculovirus-infected insect cells could deliver a high proportion of functional molecules without any need for modiWcation [33,34]. Insect cell expression should be particularly appropriate for recombinant LBDs from insect receptors, such as the ecdysone receptor. Here we describe good yields of EcR–USP LBD heterodimers produced in insect cells as recombinant DE/F segments, without fusion partners or other modiWcations. All Wve of the recombinant heterodimers expressed in this study were functional in terms of ponasterone A binding. Binding assays of cell extracts indicated that our highest expression level was equivalent to 16.6 mg active LBD heterodimer per liter of culture. Our yields of IMAC-puriWed protein ranged 2.9–16.3 mg/L culture over 10 fermentations, with a median value of 7.6 mg/L culture. Yields of 0.2–0.5 mg/L culture were reported for the vitamin D receptor LBD puriWed from recombinant insect cells [34]. The yields published for LBDs puriWed (with dimerization partners, where appropriate) from recombinant bacteria range 0.5–17 mg/L culture [35,36], with a median value of 8 mg/L culture [10,11,27,37], although sometimes only »10% of the puriWed recombinant molecules were functional [10]. The only ecdysone receptor LBD heterodimer included in those examples had been modiWed: the F-region had been deleted from its EcR component, and a fusion partner had been provided for its USP [11]. More recently, an ecdysone receptor LBD heterodimer was expressed adequately in E. coli only if a fusion partner was supplied for the EcR LBD and/or some surface residues were mutated [16]. PuriWcation, stability and function IMAC capture via the His6-tag aVorded the easiest and most eVective puriWcation of our recombinant LBD heterodimers and, in addition, ensured the removal of free USP subunits from M. persicae preparations. Although hydrophobic interaction chromatography (HIC) was apparently very eVective elsewhere [35], we found it problematic. Instead, we obtained good puriWcations using IMAC alone

or in combination with FPLC ion exchange and/or gel Wltration chromatography. The fact that all of our recombinant heterodimers could be puriWed in the absence of ligand using a single aYnity tag means that each pair of EcR and USP LBDs must associate spontaneously and with high aYnity. In contrast, the EcR and USP subunits of D. melanogaster, and recombinant LBDs therefrom, associate only weakly in the absence of ligand [13,38]. The diVerence in behavior between the L. cuprina and D. melanogaster proteins is interesting in view of the high homology (73–81% amino acid identity; Supplementary Material, Section C) between the DE/F regions of these two dipteran receptors. Wang et al. [39] proposed that the nature of the residue homologous to Tyr611 of D. melanogaster EcR (a position in helix 10 of the LBD) aVects the stability of the heterodimerization interface and may determine the extent to which the subunit engages in ligandindependent dimerization with USP. Our results show that recombinant EcR subunits with Tyr at this position do not always require the presence of a good ligand to drive heterodimerization: M. persicae has Tyr at this position, but the ligand-free LBD heterodimer was readily puriWed using only the His6-tag on the EcR subunit. We had chosen to retain much or all of the D-segments in most of the recombinant proteins because of claims that portions of the hinge regions in some nuclear hormone receptors (including ecdysone receptors) were required for LBD heterodimerization and/or ligand binding [23–26]. However, our Wnding for H. armigera that both the E/F and DE/F recombinants were active and readily puriWed as heterodimers using only the His6-tag indicates that—for this receptor, at least—the D-regions are not essential for LBD dimerization. Indeed, current knowledge from sequence alignments and crystal structures indicates that most of the previous studies were complicated by incorrect assignment of the D/E domain boundaries to positions within the E-domains. However, we can now see [16,17,40] that one of the recombinant D. melanogaster GAL4-LBD fusion proteins expressed in yeast [25] was a true DE/F heterodimer (EcR 403-652, USP 172-508), and yet it was barely functional in terms of [3H]ponasterone A binding (<0.2% of the activity of the wild-type receptor). We were therefore pleased to Wnd that the Kd values for ponasterone A binding by our four baculovirus-expressed DE/F heterodimer preparations, which spanned the range 0.7–2.5 nM (Graham et al., in preparation), were comparable to those for full-length ecdysone receptors [21]. It has been remarked previously that wild-type ecdysone apo-receptors are inherently labile, and that they need to stabilized by loading with hormone [2] and supplemented with a carrier protein when dilute [4]. Indeed, recent X-ray crystal structures show that bound ponasterone A provides interactions that stabilize the EcR LBD from H. virescens and B. tabaci [16,17]; the buried ligand constitutes a portion of the hydrophobic core needed for the structural stability of such LBDs [41]. Accordingly, we noticed that dilute solutions of our puriWed recombinant apo-LBD heterodimers

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321

Table 4 Ligand stoichiometry values for IMAC-puriWed proteins

L. cuprina L. cuprina M. persicae B. tabaci

Rt for puriWed apo-LBD heterodimersa,b

PuriWed as apo-LBD heterodimersb,c

— 2.5 (37%) 2.3 (32%) 1.3 (13%)

— 1.3 (18%) 1.2 (15%) 0.7 (7%)

PuriWed as receptor–ligand complexes 7.8 (71%)d 1.6 (23%)e 4.1 (50%)e 1.5 (17%)e

Values represent mean pmol ponasterone A binding per g protein and, in parentheses, the inferred proportion of functional LBD heterodimer in the preparations. All values in this table are for ligand binding by DE/F heterodimers in the absence of CHAPS. a Values for IMAC-puriWed apo-LBD heterodimers, obtained by expressing the total number of ligand binding sites Rt, determined by equilibrium binding experiments (Graham et al., in preparation), as a function of the total amount of protein present. b The % activity calculations use batch-speciWc % purity values (73–95%) estimated as in Table 2a, and assume a retention eYciency of »70% for ligand– heterodimer complexes on washed glass Wber discs (see Results). c Data are from standard ligand binding assays of IMAC eluates, and make the approximation that they contained saturating ligand concentrations (see Discussion). d A complex of the recombinant heterodimer with [3H]ponasterone A (65 Ci/mol) was subjected to IMAC puriWcation and the amount of radioactivity in the IMAC eluate was expressed as a function of the amount of protein present. The % activity calculation uses a batch-speciWc purity of 90%, estimated as in Table 2a. e Values assume 60% yield of recombinant protein from the IMAC step (Table 2) and no loss of activity due to denaturation of the ligand–LBD heterodimers during puriWcation. Accordingly, the stoichiometry values were calculated by expressing 60% of the initial ligand binding capability (extrapolated from standard ligand binding assays of small-scale ligand-free cell extracts) as a function of the amount of protein yielded by large-scale IMAC puriWcations of non-radioactive ligand–LBD heterodimer complexes. The % activity calculations are based on an average purity of 80% for IMAC eluates, assume a retention eYciency of »70% for ligand–heterodimer complexes on washed glass Wber discs (see Results), and make the approximation that the ligand concentration in standard activity assays is saturating (see Discussion).

were best avoided, or else had to be stabilized by including a carrier protein (0.5 mg/mL BSA) and/or saturating them with ligand. Even with large-scale IMAC puriWcations, which did not involve dilute solutions, the activity yields of apo-LBD heterodimers (16–30%) were low compared with the 70–80% yields typical of IMAC [42,44], and this seemed to reXect a high level of LBD inactivation rather than a low yield of recombinant protein (Table 2a). Indeed, the mean speciWc activity values for L. cuprina, M. persicae and B. tabaci apo-LBD heterodimer preparations (Table 4) suggested that only 7–18% of each heterodimer population had bound ligand at 2.2 nM [3H]ponasterone A, a concentration 1.8- to 3.0-times the Kd values for this ligand (Graham et al., in preparation). Similarly, comparing the concentration of binding sites (Rt, established by equilibrium binding experiments) with the concentration of recombinant protein suggested that only 13–37% of each apo-LBD heterodimer population was functional (Table 4). Other researchers attempting recombinant ecdysone apo-receptor expression have estimated that <1% and 21–73% of the molecules in their puriWed preparations were functional [9,14,15], with 50–60% estimated for puriWed preparations of apo-LBD heterodimers [11]. In our study, we inferred functionality values of 17– 50% for proteins that had been IMAC-puriWed as ponasterone A–LBD heterodimer complexes, which on average represented a 2.4-fold increase over the corresponding values for ligand-free preparations (Table 4). Direct measurement of the ligand:LBD heterodimer stoichiometry for recombinant L. cuprina LBD heterodimer stabilized by [3H]ponasterone A during IMAC indicated that 71% of the puriWed heterodimer population was functional (Table 4). Of all our stoichiometry determinations, this one was the most robust; the result shows that puriWca-

tion of ponasterone A–LBD heterodimer complexes yielded preparations in which the majority of the heterodimer molecules contained bound ligand. To obtain the highest possible ligand:heterodimer ratios in preparations destined for crystallization trials, excess ponasterone A was routinely added at the earliest opportunity, i.e., before cell lysis, and saturating concentrations were maintained thereafter. Practical issues such as protein misfolding may preclude ratios of 1:1, and a substantial proportion of bacterially expressed nuclear hormone receptor LBD populations remained unliganded even when they were puriWed in the presence of saturating ligand concentrations [37]. The ligand binding stoichiometry of puriWed apo-LBD heterodimer preparations could be increased by including the non-denaturing detergent CHAPS in the binding assays. This eVect is discussed in more detail elsewhere (Supplementary Material, Section D). The only eVect that CHAPS appeared to have on equilibrium binding data was a modest increase in the number of ligand binding sites, hinting that it may increase the proportion of functional LBDs by rescuing ligand-incompetent conformations. CHAPS seems to aVect other recombinant nuclear hormone receptors in a similar way [33]. Purity and integrity The 75 kDa contaminant that co-puriWed in many DE/ F heterodimer preparations was identiWed as heat shock protein Hsc70 from the lepidopteran host cell line. In D. melanogaster, Hsc70 binds newly synthesized EcR in vivo and helps the subunit to fold [12]. We originally thought that the 57 kDa contaminant that co-puriWed abundantly with the H. armigera E/F heterodimer might be related to

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FKBP46, a »60 kDa immunophilin that binds to the ecdysone receptor complex in Manduca sexta [43], but it proved to be -tubulin from the host cell line. -tubulin probably has high aYnity for a surface that is much more exposed in the E/F heterodimer than in the DE/F form, so the interaction is unlikely have physiological relevance. It was common for a proportion of the recombinant subunits to undergo proteolytic nicking near their C-termini. In particular, L. cuprina EcR has a long C-terminal extension (F-domain) that is absent from M. persicae EcR and—by analogy with the long and non-essential C-terminal extension in Drosophila EcR [11]—may be susceptible to clipping. The major (i.e., long N-terminal) fragments of the recombinant LBDs co-eluted with intact LBD heterodimers during non-denaturing chromatography in a way that indicated they were still heterodimerized. Although less prevalent, N-terminal proteolysis could also occur. For example, during prolonged crystallization trials of the B. tabaci ligand–LBD heterodimer complex, the D-domain and the Wrst half of helix H1 were lost from the N-terminus of the recombinant USP subunit [17]. Such trimming of poorly structured terminal regions probably aided the formation of crystals [17].

the various Cys adducts in the gel: 0 (iodoacetamidemodiWed), ¡0.5 (unalkylated), or ¡1.0 (iodoacetic acidmodiWed). A more detailed explanation of these comments is presented elsewhere (Supplementary Material, Section I) In non-denaturing IEF gels, unalkylated LBD heterodimers focused in the range pH 6.2–7.1 (Table 3). For each insect species the recombinant protein was divided more or less equally between 4 or 5 closely related isoforms, but the cause of the heterogeneity is unclear. The IEF isoforms may arise from a set of discrete conformational options being available to the D-domain, an idea consistent with the observation that the H. armigera E/F heterodimer displayed much less isoelectric heterogeneity than its DE/F counterpart (Fig. 5a). Once again, the relative mobility of alkylated LBD heterodimers (Fig. 5b) reXects the charge associated with the various Cys adducts at their Wnal positions in the immobilized pH gradient. A more detailed explanation of these comments is presented elsewhere (Supplementary Material, Section I). Conclusion and relevance

In non-reducing conditions 74 °C, disulWde bonds formed over a period of days within and between some types of recombinant LBD (for a detailed discussion , see Supplementary Material, Section H). Recombinant USP did not seem to engage in intermolecular bonding, whereas most if not all of the EcR did. The process seemed to be non-speciWc, and probably involved Cys residues located in relatively unstructured regions (D-, and possibly F-segments). Although this source of structural heterogeneity could be prevented by alkylation of the proteins, such treatments introduced chemical microheterogeneity (Supplementary Material, Section E (b)) that was itself likely to hinder crystal formation. In fact, our best crystallization results were obtained when unalkylated proteins were maintained in reducing environments [17].

Our results show that neither ligand nor D-regions are essential for the formation of tightly associated and functional ecdysone receptor LBD heterodimers, and indicate that the presence of ligand enhances heterodimer stability. The puriWed preparations of DE/F apo-LBD heterodimers display ecdysteroid-binding aYnities that are similar to those of full-length ecdysone receptors, so they can be used to screen chemical libraries for new agonists and antagonists and to investigate in vitro the selectivity of ligands across taxonomic orders (Graham et al., in preparation). CHAPS may enhance the sensitivity of such assays. Recombinant apo-LBD heterodimer preparations have already enabled us to show that the binding aYnities observed in vitro for the bisacylhydrazine ligand RH5992 reXect the insecticidal activity of this compound in vivo [17]. PuriWed preparations of the B. tabaci ponasterone A–LBD heterodimer complex gave crystals that aVorded the Wrst atomic structure of an LBD heterodimer from a hemipteran ecdysone receptor [17].

Non-denaturing gels

Acknowledgments

Native gel electrophoresis reveals both non-covalent and covalent assemblies, and both types seem to be present here (Fig. 4). The fact that [3H]ponasterone A comigrated speciWcally with each of the recombinant bands in our native gels, and did so in proportion to the amount of protein present (Fig. 4a), indicates that the recombinant LBD heterodimer and its oligomers were functional. The smaller size (Table 1) and lower pI (Table 3) of the M. persicae LBD heterodimer are both likely to contribute to its higher mobility relative to the L. cuprina one (Fig. 4c). The relative mobility of alkylated LBD heterodimers (Fig. 4b) simply reXects the charge associated with

This work was supported by an AusIndustry START award (1999–2002). We are grateful for assistance early in the project from Hieu Nguyen (SDS–PAGE and assays), and from Neva Ivancic and Miriam Eldridge (recombinant insect cell fermentations). We thank Mike Lawrence for helpful discussions.

DisulWde bond formation

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.pep. 2006.12.011.

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