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Molecular Characterization by Mass Spectrometry of the Human Estrogen Receptor Ligand-Binding Domain Expressed in Escherichia co/i Donald A. Seielstad, Kathryn E. Carlson, John A. Katzenellenbogen, Peter J. Kushner, and Geoffrey L. Greene Department of Chemistry University of Illinois Urbana, Illinois 61801 (D.A.% K.E.C., J.A.K.) Metabolic Research Unit (P.J.K.) University of California San Francisco, California 94143 Ben May Institute (G.L.G.) University of Chicago Chicago, Illinois 60637 The ligand binding domain of the human estrogen receptor (hER-LBD), encompassing the sequence MDP!$,&G . . . V,,, has been expressed at high levels in Escherichia co/i from a PET-23d vector, and a purified preparation has been characterized both by mass spectrometry and biochemical methods. Inclusion bodies from the bacterial expression were solubilized by sonication and the hER-LBD was purified to near homogeneity by affinity chromatography over an estradiol-Sepharose column in urea-containing buffer. This material ran as a single peak on reversed-phase HPLC, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed a band with apparent molecular mass of 31-32 kilodaltons (kDa), somewhat smaller than that expected from the construct (36.6 kDa). Edman degradation revealed a single sequence of MDPSAGDMRA, consistent with an intact N terminus. Further characterization of this material using low resolution matrix-assisted laser desorption ionization mass spectrometry indicated an apparent single protein species of average mass 33,143 daltons (Da). Detailed molecular analysis by electrospray ionization mass spectrometry, however, revealed that this purified hEl?LBD preparation was actually composed of a number of both modified and unmodified LBD protein fragments between 32$00-33,400 Da. The bulk of this 33-kDa protein mixture consisted of three LBD protein fragments with C termini at Ala,, (70%), Ala, (23%), and Ser,,, (4%), with only a trace amount (3%) of the full length expressed LBD (MDPS, . . . V-; 35, 602 Da). These four protein species also appear to be partially chemically modified by carbamylation. The functional integrity of this hER-LBD preparation was investigated by a ligand-exchange assay, which showed that the hER-LBD retained full estradiol-binding capacity; specific, covalent labeling was also observed using either the electrophilic affinity-labeling ligand tamoxifen aziridine (TAZ) or the photoaffinity-labeling ligand hexestrol diazirine. Thus, this expressed hER-LBD preparation, while appearing nominally pure by conventional biochemical techniques and having the expected ligand-binding capacity, was shown by sensitive high resolution electrospray ionization mass spectrometty techniques to be largely truncated 20-26 amino acids from the expected C terminus and to have a substantial level of covalent modification arising from the urea. Our findings highlight the power of mass spectrometric analysis to identify and to characterize, at the molecular level, the heterogeneity that may be present in expressed receptor preparations that appear nominally pure by conventional methods of analysis. Such information on the sites and nature of cleavage and modification is important in devising appropriate expression systems and purification protocols and can serve as a specific guide for obtaining preparations of the hER-LBD that are homogenous and potentially better suited for nuclear magnetic resonance and x-ray crystallographic analysis. (Molecular Endocrinology 9: 647-666, 1995) 0888-8809~9563.00~0 Molecular Endocrinology Copyright 0 1995 by The Endocrine Society 647 MOL 648 END0 . 1995 INTRODUCTION The estrogen receptor (ER) is a ligand-activated generegulatory protein that is a member of the nuclear receptor superfamily (l-4). Like the other members of this superfamily, ER is a multidomain protein, having distinct amino acid sequences associated with the major functions of DNA interaction (domain C) and ligand binding (domain E) (l-4). While the functional characteristics of these domains have been probed extensively by mutagenesis methods, direct structural characterization has been rather limited. The DNA binding domains of these nuclear receptors are small, polar, highly conserved regions consisting of 65-85 amino acids and have been readily expressed in a number of heterologous systems. Additionally, the ER, glucocorticoid receptor, and retinoic acid receptor DNA binding domains have been the subject of a number of successful x-ray and nuclear magnetic resonance (NMR) structural studies and have been shown to exist in a helix-loop-helix topology in which a zinc atom chelated by four cysteines stabilizes the N terminus of each helix (5-l 0). The ligand binding domains (domain E) of the nuclear receptors, by contrast, are much longer (- 250 amino acids), and while they have been expressed efficiently in a number of systems (1 l-13), their hydrophobic character has limited their solubility in aqueous systems. Consequently, the generation of stable crystals of the ligand-binding domain suitable for x-ray analysis has proven to be difficult, and their low solubility has limited analysis by NMR. It is becoming evident that conventional means for characterizing protein purity and identity [sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDSPAGE), reversed-phase (RP)-HPLC, N-terminal Edman sequencing], and functional characterization (ligand binding, immunological assays, and affinity labeling), are insufficient to specify the precise molecular species that have been produced by and purified from heterologous expression systems (14, 15). There are examples where heterologously expressed proteins have not been fully translated, or have been proteolyzed, and are thus mixtures of closely related species (1 S), have lacked important posttranslational modifications, or have been subject to oxidation or other inadvertent chemical derivatizations (17-l 9). In some cases, these changes have altered the properties of these expressed proteins sufficiently so as to interfere with structural studies by x-ray and NMR analysis (16-19). The recently developed mass spectrometry methods of matrix-assisted laser desorption ionization (MALDI-MS) and electrospray ionization mass spectrometry (ESI-MS) afford new opportunities for the characterization of high molecular weight proteins, to ascertain both their identity and their purity (14-20). In this report we describe the development of a high yield expression system for the production of the ligand-binding domain of the human estrogen receptor Vol 9 No. 6 (hER-LBD) in Escherichia coli and a two-step scheme for the solubilization and purification of this species in a form that binds the ligand estradiol (E.-J. We have also characterized this species by both conventional methods (ligand binding, affinity labeling, SDS-PAGE analysis, and Edman sequencing), as well as by MALDI-MS and ESI-MS. Through application of HPLC/ ESI-MS methods, we have identified unexpected structural and chemical alterations in the hER-LBD that have arisen through the expression and purification procedures employed. Since the nature of these alterations can be ascertained by ESI-MS, this information can serve as a guide for obtaining homogenous preparations of the hER-LBD. RESULTS Plasmid Construction, E. co/i, and Purification hER-LBD Expression in Initial attempts to express the full length hER in bacteria suggested that expression was relatively low, as had been reported by others (21). Consequently, we constructed a series of expression vectors in which the ligand binding domain (LBD) and part of the hinge region (amino acids 282-595) could be expressed in E. co/i. These LBD vectors, in which the ER domain is fused to a polyhistidine region, to a short peptide encoding an epitope from phage T7 Gene 10, or to a three-amino acid lead sequence (MDP, the PET-23dERG vector) are illustrated in Fig. 1. Expression of each of these vectors in E. co/i was equally robust. The recombinant protein was a significant fraction (2030%) of all the cellular proteins, as revealed by SDSPAGE analysis of total cell lysates (not shown, but see below), and amounted to between 60 and 90 mg/liter of bacterial culture. A procedure to isolate and purify the expression product from the PET-23d-ERG vector, encoding the sequence MDPS,,, . , . V,,, was developed. After cell growth and protein induction under optimized conditions, we found the bulk of the hER-LBD in the cell pellet after sonic lysis, perhaps trapped in inclusion bodies. Proteins in the pellet fraction were solubilized by repeated sonication in 1 M urea-containing buffer. SDS-PAGE analysis of the soluble extract (Fig. 2, lane 1) reveals that the hER-LBD is BamH I BamH I CCATGGATCCATC6 - MetAspPrO~~2;2- Fig. 1. A Schematic Showing in the PET-23d-ERG Vector For details, see text. the - - - - - GTC - - - - - "&5,, hER,,,-,,, Insertion Site Human ER Ligand Binding kDa 649 Domain 12 il.00 31- 0.25 BOUND 0.50 0.75 13H] ESTRADIOL Fig. 3. Scatchard Analysis of the Capacity of the Solubilized Bacterial sion of the PET-23d-ERG Vector Assay was done after a 2300-fold text. Analysis of Size and Purity SDS-PAGE Fig. 2. SDS-PAGE Analysis of hER-LBD Purification hEf?-LBD was purified from bacterial extract by specific adsorption onto E,-Sepharose and elution with reduced specific activity rH]E,. Bacterial extract (lane 1) and eluted proteins (lane 2) were analyzed by SDS-PAGE on a 12% gel, and proteins were visualized by staining with silver (22). A major protein band was observed at 31-32 kDa and a minor band at the expected 35 kDa. Each lane contained approximately 10 pmol ER-LBD, as determined by assay on controlled porosity glass. Migration of molecular weight markers is indicated at the left. For details, see text. the most abundant protein. Scatchard analysis of the E,-binding activity in this solubilized extract, after appropriate dilution, indicated an equilibrium dissociation constant (Kd) of 0.29 nM (Fig. 3), which is consistent with values obtained for full length hER (13, 21-23) although somewhat above that for an expressed fragment of hER containing the LBD, [which was assayed at higher dilution (24)]. Purification of the hER-LBD was effected in one step, by increasing the urea concentration to 3 M, adsorbing the hER-LBD onto an E,-Sepharose column (25,26), washing the column with 5 M urea, and eluting it with excess low specific activity E, in 5 M urea. In this manner, the hER-LBD was obtained as a nearly homogenous species (Fig. 2, lane 2). Comparison of the amount of protein recovered from the affinity column with that expected on the basis of the binding assay in the solubilized extract indicated that the purification process was essentially quantitative. Furthermore, by proteinand ligand-binding assays, this purified hER-LBD binds one equivalent of E,. 1 .oo E, Binding Cell Pellet, dilution. 1.25 (nM) Affinity and after Expres- For details, of the hER-LBD see by When assayed for size and purity on SDS-PAGE, the great majority of the hER-LBD migrated as a single band with an apparent mass of 3132 kDa (Fig. 2, lane 2). Only a very minor fraction was observed to migrate as the 35.6-kDa species expected from the hER-LBD construct. No other protein bands were visible, even when the gel was purposefully overloaded and the proteins detected with a sensitive silver stain (27). Because of the construction of the PET-23d-ERG expression plasmid, the expressed hER-LBD begins at residue position Ser2** of the ER sequence and is preceded by a sequence of three amino acids that differ from wild type hER. The region (amino acids 299-303) generally considered to be the start of the ligand binding domain (E) (l-4, 28-30), has been characterized as being proteolytically sensitive (31, 32). :-tad the hER-LBD been fully expressed to the C terminus then subsequently clipped at this site, it would have had a molecular mass of 32,800 daltons (Da) to 33,300 Da, more consistent with that observed by SDS-PAGE (31-32 kDa). N-terminal analysis by the Edman method, however, showed a clear, single sequence of MDPSAGDMRA, indicating that the N terminus was intact. The composition of the C terminus was elucidated by ESI-MS, as discussed below. Analysis of hER-LBD by RP-HPLC RP-HPLC is an effective method for analyzing the of picomole quantities of proteins. A typical HPLC UV chromatogram of the hER-LBD receptor preparation determined at 280 nm is shown in Fig. 4. The broad, off-scale peak that starts with the solvent front and continues for some 20 min is attributed purity MOL END0 Vol 9 No. 6 . 1995 Analysis 6 4 2 0 ~ 0 50 100 Retention 150 Time (min) 200 250 Fig. 4. RP-HPLC Trace of the hER-LBD Preparation Purified ER preparation (150 pmol) was injected into a 250 mm x 2.1 mm C-4 column and eluted with the compound gradient of (H,O, 0.06% CF,CO,H) and (CH,CN, 0.05% CF,CO,H) (see Materials and Methods). The eluant was monitored at 280 nm; the offscale peak that follows the solvent front is urea and buffer salts; the peak at 85 min is rH]E,, and the peak at 114.5 min is the hER-LBD. and other buffer salts. The strong peak at 84.3 min is identified as free E,; this was confirmed by subsequent injection of rH]E, under identical conditions. The peak observed at 114.5 min, corresponding to approximately 72% CH,CN, is attributed to the hER-LBD. Protein assay showed that this fraction did indeed contain protein, and SDS-PAGE showed the same 3132 kDa band present in the original preparation. Thus, by both SDS-PAGE and HPLC analysis, the expressed hER-LBD appears to be largely a single protein species. to urea Analysis of hER-LBD by MALDI-MS While MALDI-MS has higher sensitivity than other mass spectroscopic techniques and has a higher mass range, it does so at the cost of resolution (14, 33). Consequently, mixtures of large biomolecules differing by 1 O-200 Da are difficult to resolve. Matrices of the hER-LBD, prepared by an alternate method (34) because of the high urea in the sample, were subjected to MALDI-MS and provided very good signals for [M+H]+‘, [M+2H]+‘, and [M-I-~H]+~ ions, indicating an average molecular mass for the major receptor component of 33,143 Da, as shown in Fig. 5. This mass estimate is quite consistent with both the SDSPAGE (3132 kDa) and ESI-MS determinations (33,093) (see below). of hER-LBD by ESI-MS Since the ESI-MS cannot tolerate appreciable salt concentrations, a microbore HPLC system coupled to a LC/ESI-MS interface (see above) was used to desalt and remove the 5 M urea present in the hER-LBD preparation (see Materials and Methods). Typical injections contained 150 pmol receptor (5 ~1 of a 30 pmol/pl solution in 5 M urea) and used somewhat steeper acetonitrile elution gradients than shown in Fig. 4 above; under these conditions, hER-LBD elutes at 57 min. Inspection of the raw ESI-MS data for this peak (Fig. 6) indicates that there are at least three major multiply charged ion series present in the HPLC eluate at 57 min. Deconvolution of these ion series using maximum entropy analysis, shown in Fig. 7, identified three major series of modified proteins at 32,923 Da, 33,093 Da, and 33,423 Da, as well as a trace amount of protein with molecular mass of 35,595 Da; an expansion of the major protein components between 32.833.5 kDa is shown as the inset in Fig. 7. The ESI-MS technique provides mass accuracies up to 0.05%, so these 33-kDa proteins can be expected to have errors 2 17 Da. The expression system was designed to produce the hER-LBD protein MDPS282-V595r which would be expected to have a molecular mass of 35,604 Da. N-Terminal sequencing via Edman degradation of the protein mixture provided only the N-terminal residues expected for this construct (see above). The presence of a small amount of the full-length receptor sequence in the purified preparation, as indicated by the 35,595-Da peak by ESI-MS, confirmed that the DNA coding sequence in this expression system is properly constituted. Analysis using either Masslynx BioAnalysis or MacBioSpec (v.1 .O.l , Perkin-Elmer SCIEX Division, Thornhill, Ontario, Canada) suggested C-terminal truncations compatible with the three other protein masses detected by ESI-MS; these are shown in Table 1. C-termini are proposed at Ala,,,, Ala,,, , and Ser,,,. Close inspection of the major zero-charge molecular mass peaks observed by ESI-MS (Fig. 7, inset, especially the 33,093-Da peak) revealed the presence of an additional series of masses at [M+n*(43)], where n = 0,1,2,3. This suggests that each component protein has been covalently modified in some manner, either before or during analysis. The most likely modification would be carbamylation, which should manifest itself as a series of [Ml, [M+43], [M+86], [M+129]. In this hER-LBD preparation, the extent of monocarbamylation and dicarbamylation is estimated at 22% and 7%, respectively. Carbamylation could result from the reaction of the lysine E-amino group or the cysteine thiol group with cyanate ion, which is generated as an equilibrium product from the high concentrations of urea used for the isolation and purification of the hERLBD (35, 36). The presence of the various mass components observed by ESI-MS indicates that a large proportion of the proteins in the hER-LBD preparation Human ER Ligand Binding 651 Domain 100 1.5E3 60 m/z Fig. 5. The MALDI-MS of the hER-LBD This is a spectrum of a sample applied to the probe by a protocol that allows the excess urea to be removed by gentle (see Materials and Methods). Visible are peaks due to the singly charged and doubly charged species, as the average of 33,143 and 16,604 Da, respectively. 10( washing masses oqa.5 !.l I 182.7 8 895. a00 6. ESI-MS of the hER-LBD This spectrum shows several left, each envelope results from 900 10 Fig. are either truncated (within terminus) or are chemically or both. Characterization of the hER-LBD envelopes of peaks that progressively increasing correspond to species charge on each mass -26 amino acids of the C modified in some manner, of the Ligand-Binding Capacity The ability of hER-LBD to bind E,, even in the presence of 5 M urea, is implied because it was bound by of different fragment, mass which (cf. Fig. 7). Going from lowers its m/z value. right to an E, affinity column under these conditions, and it elutes in 5 M urea only in the presence of E,; in addition, the eluted hER-LBD is bound to one equivalent of low specific activity (0.25 CVmmol) rH]E,, as determined by specific adsorption of hER-LBD-rH]E, complex to controlled-pore glass (CPG) beads (see Materials and Methods). Because the purified hER-LBD is already occupied by ligand, standard protocols cannot be used to determine its binding affinity. We have, MOL END0 . 1995 Vol 9 No. 6 652 101 7 33093.3 33200 33600 14000 mass 3366.6 34400 34600 35200 356( 35594.6 ALI ‘200 33600 34000 34400 34600 35200 35600 mass Fig. 7. The Deconvolution of the ES-MS of the hER-LBD, Shown in Fig. 6 Main panel shows the spectrum of zero charge mass from 32,800 to 35,800 Da; inset is an expansion of the 35,800 to 33,550 Da region. The four different length species in the hER-LBD preparation give their primary peaks at 32,923, 33,093, 33,423, and 35,595 Da, respectively. The monoand dicarbamylated species, in each case, appear as smaller peaks at intervals of approximately 43 and 86 Da higher mass. These are especially apparent with the most intense species (33,093). The deconvolution routine does not always give clear, single peaks, especially for the peaks with weaker intensities. however, been able to confirm further, by exchange assays with high specific activity pH]E,, that most of the purified hER-LBD is active in binding E,. For example, with 2 mg/ml BSA added as carrier protein, 2000-fold diluted hER-LBD was allowed to exchange at either 0 C or 25 C and assayed by hydroxylapatite (HAP) adsorption (37). Little exchange was observed at 0 C: 4% after 1 h, which increased slowly to 26% after 24 h (data not shown). Exchange proceeded quickly at 25 C, however, reaching 49% after 1 h and 84-94% after 4 h. It remained constant and stable from 4-24 h (Fig. 8). These kinetics are consistent with E, exchange rates measured with rat uterine ER (38). Greater than 90% of this exchange could be blocked by the addition of excess unlabeled E, (Fig. 8); no specific rH]E, binding was found with the carrier BSA alone. While the hER-LBD will undergo exchange in 5 M urea, with or without dilution, the diluted form is less stable under these conditions. The addition of BSA carrier protein is not essential, but it reduces adsorption losses of diluted preparations. Using a dye-binding assay (39), we determined that the hER-LBD preparation (before dilution and carrier protein addition) had a protein concentration of 1.03 mg/ml, which makes it 3.11 X lop5 M, assuming a molecular mass of 33,100 Da. The E,-binding capacity measured by this exchange assay was 2.61-2.94 x 10m5 M, which is, again, consistent with a 1:l binding stoichiometry, the same as that found directly after elution from the E, affinity column. Thus, according to standard exchange binding assays, the hER-LBD-purified preparation has the expected capacity for binding E,. As noted earlier (Fig. 3), Scatchard analysis of the unoccupied hER-LBD, solubilized in 1 M urea before purification by affinity chromatography, indicated that it had a dissociation constant (Kd) of 0.29 nM. Affinity Labeling of hER-LBD with TAZ and Hexestrol Diazirine The well characterized electrophilic affinity label, TAZ, can be exchanged into the ligand-binding site of the hER-LBD. TAZ (40) is an efficient and selective electrophilic affinity-labeling agent for unoccupied ER, but its rapid action [>90% of the reactive aziridine labeling moiety of TAZ is consumed within the first 0.5 h (40)] makes it less efficient under the exchange conditions needed to label the hER-LBD. Nevertheless, TAZ is able to label 38% of the hER-LBD (data not shown). Photoaffinity ligands behave as reversible ligands until they are exposed to light. Therefore, they can be incubated with the hER-LBD under exchange conditions for several hours in the dark so as to undergo near-maximal exchange, whereupon brief photolysis can effect covalent attachment of the ligand. Hexestrol diazirine, a new photoaffinity labeling agent (41), binds reversibly to the hER-LBD after exchange; photolysis then effects covalent attachment to 38% of the total sites (or 70% of the sites occupied after exchange; data not shown), which is comparable to that achieved Human ER Ligand TABLE 1. Major Binding Protein Fragments Receptor MDPS,,,AG MDPS,,,AG MDPS,,,AG MDPS,,AG . . . . . . . . N Il. Il. Il. Domain 653 Observed In HPLWESCMS fragment . ITGEAEGFPATV,,, . DQSHLATAGSTS,,, . . DQSHLAT&,, . DQSHLA,,, with full length ER from rat uterus (41). The attachment of both reagents is stable to the conditions of SDSPAGE and showed radioactive bands that comigrated with the 3132 kDa species (not shown). DISCUSSION The development of methods for the high-level expression of proteins in heterologous systems and their purification in homogenous, functional form are prerequisites for detailed studies of their structure by spectroscopic methods. In this report, we have described the development of an expression system for the LBD of the hER (MDPS,,,-V,,5) in f. co/i and a simple and efficient purification of this receptor domain by chromatography on an E, affinity column in 3-5 M urea after solubilization in 1 M urea. The purified hER-LBD was characterized with respect to size, purity, and ligand-binding capacity using a variety of classical biochemical techniques, as well as newly developed mass spectrometry methods. The latter methods revealed an unexpected heterogeneity in this preparation, the nature of which has led to a redesign of the system for expression and purification of the hER-LBD. 1001 I ___-_-_-----_---_-_. ------0’ .. .- ___ *’___________ 10 15 20 25 0 5 HOURSOFEXCHANGE Fig. 6. Time Course of Exchange of High Specific Activity PH]E, into the binding site of the hER-LBD at 25 C. The hER-LBD was diluted to 10 nM ER with BSA added as carrier protein. This was incubated with 43 nM rH]E, (total) z-1 p,~ unlabeled E, (blocked). Exchange was measured by the HAP assay and is expressed as a percent of the total hER-LBD. Specific exchange (bold line) is the difference between total (solid line) and blocked (dashed line). Analysis of Original Receptor Mass (Da) (predicted) Mass (Da) (observed) 35603.6 33522.2 33089.9 32917.7 35594.6 33422.7 33093.3 32923.3 Preparation (dacns) 9.0 0.5 3.4 5.6 Relative abundance 3% 4% 70% 23% By conventional techniques of SDS-PAGE, HPLC, and Edman N-terminal sequencing, the purified hERLBD appeared to be an essentially homogenous protein component of 32 kDa, with a single, intact N terminus. Although the hER-LBD migrated on SDSPAGE as a somewhat smaller mass than that expected for the expressed protein (35.6 kDa), this could be attributed to variations in SDS association due to the highly hydrophobic nature of the LBD. The intact N terminus indicated that cleavage had not occurred at the known proteolysis-sensitive sites in the region of amino acids 299-303. Both before and after its purification by E,-Sepharose affinity chromatography, the hER-LBD was shown to bind E,. The eluted, purified protein displayed the expected 1 :l ligand-binding stoichiometry both by direct binding and by exchange, and the photoaffinity label, hexestrol diazirine, underwent covalent attachment to the hER-LBD with an efficiency nearly comparable to that obtained with fulllength ER. These studies indicate that the hER-LBD, by itself, has the tertiary structure needed for high affinity ligand binding. When the LBD was examined by the recently developed mass spectrometry method of MALDI-MS, the preparation appeared to be a homogenous protein of molecular mass 33,143 Da, consistent with the estimate by SDS-PAGE. RP-HPLC, which is frequently used to characterize the homogeneity of protein preparations, showed the purified hER-LBD as a single protein peak, eluting at high CH,CN concentration (72%). However, when this technique was coupled to ESI-MS, the microcomposition of the hER-LBD eluate was revealed. This high resolution mass spectrometry method showed that the hER-LBD preparation consisted of four components sharing a common N terminus, but having various C termini: full-length hERLBD ending at Valsss, and three fragments truncated at Ser,,,, Ala,,, , and Ala,,,. Their observed molecular masses (and relative abundances) were 35,595 Da (3%), 33,423 Da (4%), 33,093 Da (70%), and 32,923 Da (23%), respectively. These truncations could arise either from incomplete translation or from physical or enzymatic cleavage of the intact hER-LBD. The occasional presence of codons in eukaryotic genes that are rarely used in prokaryotes does not usually result in premature truncation, although clusters of rarely used codons may account for reduced expression yields (42, 43). Of the nucleotide sequences that code for the amino acid pairs spanning the three observed premature termina- MOL END0 1995 Vol 9 No. 6 654 tion sites: Ala,,,-Thr,,, (GCC-ACU), Ala57,-Gly572 (GCG-GGC), and Ser,,,-Ser,,G (UCA-UCG), only the Ser UCG codon is infrequently used in f. co/i genes (44, 45); however, it is also infrequently used in human genes, and this Ser-Ser sequence does not correspond to the predominant site of cleavage, which is at Ab,. Although physical cleavage of the hER-LBD during its solubilization by sonication is possible, this is not a well documented phenomenon. C-Terminal truncation of expressed proteins by proteolysis is well known; however, the sequences at the three C-terminal truncation sites do not correspond to the substrate specificity of bacterial proteases most commonly implicated in the degradation of heterologously expressed proteins (46-48). The sequence in the region of the hER-LBD that suffers cleavage at these three sites is unusual, being rich in serine and threonine residues, and it bears considerable similarity to signal peptide leader sequences found at the N termini of proteins that are directed to the periplasmic space (e.g. ompT). In E. cob, these leader sequences are cleaved by a recently characterized leader peptidase (SPase I, 49), which has a preference for small residues at the P, and P, sites, and a helix-breaking residue (Gly, Pro, or Ser) at the P,-, sites. The sequence in this region fulfills this motif at all of the three cleavage sites (indicated by a co/on) and would account for the predominant cleavage at As7,: Dse4 Q S H L A:T A:G S T S:S,,, The signal leader peptidase is an integral inner membrane protein that normally cleaves the N-terminal signal sequence of proteins during their secretion into the periplasmic space. Thus, this peptidase would cofractionate with the hER-LBD in the pellet fraction after sonic lysis of the cells, and it may be cleaving the LBD at the three C-terminal cryptic cleavage sites during the solubilization process that involves repeated sonication in 1 M urea. This supposition is supported by the observation that hER-LBD that is solubilized in the initial rounds of sonication contains a greater amount of the 35kDa species (the size corresponding to the intact LBD), while the final phases of solubilization release material that is almost exclusively 3132 kDa (G.L. Greene, unpublished). As the signal leader peptidase is very difficult to inhibit (49), it is unlikely that this cleavage can be entirely blocked. Analysis of the hER-HBD by ESI-MS also revealed that all four length components were chemically modified by mono- and dicarbamylation to the extent of 22% and 7%, respectively. It is known that cyanate ion, generated via a slow equilibrium reaction from aqueous urea solutions and therefore often present as a contaminant, can readily carbamylate cysteine y-thiol and lysine E-amino side chains (35, 36). Therefore, although appearing to be homogenous and functionally active, in terms of E,-binding capacity and affinity-labeling specificity, this hER-LBD preparation is, in fact, a mixture of peptides truncated adventitiously 20-26 residues from the C terminus, with significant modification by carbamylation. It is known that microheterogeneity of heterologously expressed proteins can affect solubility and can interfere with crystallization, thereby complicating structural studies by x-ray and NMR (16-l 9). Therefore, it is essential to fully characterize the purity and the identity of expressed proteins at the molecular level. Conventional methods for ascertaining purity and identity, such as SDS-PAGE, Edman N-terminal sequencing, and HPLC, can reveal major structural anomalies, but are unable to detect variations in microstructure; by contrast, molecular composition is readily disclosed by the more sensitive and higher resolution mass spectrometry technique ESI-MS. A detailed knowledge of chemical constitution at this level may be critical in designing and obtaining expressed preparations of steroid receptors that are suitable for characterization by NMR and x-ray crystallographic analysis. In fact, on the basis of the specific information on the C-terminal cleavage and carbamylation of the hER-LBD preparation that we analyzed in this report, we have redesigned the expression construct so as not to include the C-terminal cleaved region (which is actually in the F-domain), and we have modified the purification protocol to minimize exposure of the LBD to cyanate. As will be described in full elsewhere, this revised expression and purification protocol results in an hER-LBD preparation that now appears to be homogeneous and functional by all of the criteria used in this report. MATERIALS AND METHODS Materials Radioligands were obtained from the following sources: vH]E, ([6, 7-3H]estra-l, 3, 5,(10)-triene-3,176-diol) 51-53 Ci/ mmol; PH]TAZ ([ring-3H]-(Z)-[4-[2-(N-aziridinyl) ethoxylphenyl]]-1,2-diphenyl-1 -butene), 20 Ci/mmol from Amersham Coro (Arlinaton Heiahts. IL): f Hlhexestrol diazirine (PHl-3azidutyl 2, s-bis(4-h~droxyphenyljpentyl sulfide), 32 CGmmol prepared in our laboratory (41); unlabeled ligands: E, from Sigma; TAZ (40) and hexestrol diazirine (41) were prepared in our laboratory as previously reported. The following compounds were obtained from the sources indicated: HAP and Kaleidoscope prestained mol wt standards (Bio-Rad, Hercules CA); CPG beads (500 A pore size; Electra-Nucleonics, Inc., Fairfield, NJ); pET‘vecto&, E. co/i strains, and growth broth (Novagen Inc., Madison WI). The E,-Sepharose (ESeph) affinity column was prepared as previously reported (25). Construction of Expression Vectors for hEF?-LBD To construct a fusion protein of the ER-LBD to a leader with an epitope from phage T7 Gene 10 or to a polyhistidine leader, a BarnHI-EcoRI fragment spanning the domain (S,,,V,,,) from HE 14 (50) was inserted into PET-1 7b and PET-1 9b vectors (Novagen Inc.), respectively. These constructions incorporated the Val,,, mutation present in the original ER cDNA (51) but were converted to the wild type Gly,,, by substitution of an Ncol-EcoRI fragment or a HindIll-EcoRI fragment, respectively, from pCMV-ER [a gift from Dr. Benita S. Katzenellenbogen (52,53)]. To construct a fusion protein of the ER sequences to the three-amino acid leader (MDP), an Ncol-EcoRI fragment spanning the C-terminal portion of the Human ER Ligand Binding 655 Domain domain from pCMV-ER was inserted into a PET-23d vector (Novagen Inc.). An Ncol fragment incorporating the leader sequences and amino-terminal portion from HE 14 were subsequently inserted to complete the construction of PET-23dERG expression vector. This plasmid contains the sequence from the carboxy-terminal region of wild type Sebdhg5 hER, preceded by an MDP sequence, and has Gly at position 400 (MDPS,,,-V,,,). Expression and Purification of hER-LBD Conditions suggested in the manual provided by the makers of the PET vectors (Novagen Inc.) for cell growth and protein induction were followed precisely, except that medium was 2x LB. hER-LBD was overexpressed in BL21 (DE3 A lysogen) cells transformed with the PET-23d-ERG vector. Cells were grown in selective medium (2x LB + ampicillin) to an absorbance of 2.0 (@ 600 nm) and then induced with 0.6 mM isopropyl+-thiogalactopyranoside for 3 h at room temperature (23 C). Cells were pelleted by centrifugation, snap frozen, and stored at -75 C. Each liter of culture yielded a 5-ml pellet. Frozen cells were thawed and lysed by sonication (4 x 20 set) in four volumes (20 ml) of lysis buffer [50 mM Tris; 50 mM NaCI. 1 mM EDTA. 1 mM dithiothreitol (D7-n. 1 M urea. oH 7.41. This procedure ‘was repeated several tknes for each pellet. Clarified fractions were obtained by centrifugation at 30,000 x g for 30 min, and 61 ml pooled extracts were adjusted to contain 3 M urea and 0.4 M NaCl (loading buffer) and then applied to a 20-ml column of E,-Sepharose (2526). The column was washed successively with loading buffer (240 ml) and loading buffer that contained 5 M urea (50 ml). Bound hER-LBD was eluted with 2 x 1 O-5 M rH]E, (specific activity 0.25 Ci/mmol) in 30 ml of a buffer that contained 25 mM Tris, 100 mM NaCI, 1 mM EDTA, 1 mM DlT, and 5 M urea, pH 7.4. The yield of hER-LBD, as determined by specific adsorption of bound PH]E, to CPG beads (54) was 156 nmol (100% recovery). The protein content, as determined by the method of Bearden (39) with BSA as the protein standard, was 4.9 mg, which agreed closely with the predicted yield of 5.0 mg for a determined hER-LBD mass of 33 kDa. hER-LBD purity was also assessed on silver-stained SDS-polyacrylamide gels and by Western blot analysis with the H222 monoclonal rat antibody (55, 56). The major eluate fraction from the E,-Sepharose column was supplemented with 5% glycerol and concentrated by centrifugation through an Amicon Centriprep-10 filter to a final volume of 3.5 ml and an hER-LBD concentration of 31 nmol/ml [31.3 PM, containing 62 PM E, (0.25 Ci/mmol)]. Aliquots were snap frozen and stored at -75 C. hER-LBD Binding For exchange in the sample was routinely Assay and Affinity SDS-PAGE Cell lysate fractions and purified hER-LBD were analyzed by SDS-PAGE as described previously (25) except that a 12% reducing gel was used. Samples for N-terminal sequencing were electrophoresed using the Tricine system (59). Gels were either stained with Coomassie Blue G (59) or silver (27) for visualization or electroblotted to polyvinylidene fluoride membrane for N-terminal sequencing. Molecular weight prestained standards were from Bio-Rad. N-Terminal Microsequencing Gel patterns were electroblotted to Pro-Blott polyvinylidene fluoride membranes and stained with Coomassie Blue R, following the recommendations of the manufacturer for the Pro-Blott membrane (Applied Biosystems, Inc). Bands of interest were excised with a razor blade and N-terminal sequenced by Edman chemistry on an Applied Biosystems model 477A protein sequencer coupled to a model 120A on-line PTH analyzer by Dr. K.-L. Ngai and the personnel in the Genetic Engineering Facility of the University of Illinois. Protein Determination Total protein was assayed using Standard curves were generated Analysis The equilibrium-binding affinity of hER-LBD for [3H]E2 was determined by saturation analysis of an unpurified bacterial extract that was diluted (2300-fold) to contain approximately 1 nM unliganded hER-LBD in binding buffer (50 mM Tris, 50 mM NaCI, 1 mM EDTA, 1 mM DlT, and 0.1% ovalbumin, pH 7.4). Increasing amounts of [6,7-3H]E, (0.2-20 nM; 53 Ci/mmol) were added, and solutions were incubated overnight at 4 C. Bound [3H]E2 was determined by specific adsorption of [3H]E,-hER-LBD to CPG beads (54):Free [3H]E2 was determined by subtraction of bound f3H1E, from total added f3H1E,. Nonspecific bindina was mea&red in the presence of a 200-f&d excess of-unlabeled E,. An equilibrium dissociation constant (Kd) was calculated by the method of Scatchard (57). Exchange C), giving a final concentration of 3 nmol/ml ER and 0.5 M urea. The preparation could also be exchanged without dilution (i,e. at 31 nmol/ml ER, 5 M urea). The 3H ligand at a specific activity of 10-15 Ci/mmol was added to be 2- to 3 times the ER concentration. A control was always run in parallel where the specific exchange was blocked by a lo-fold, or greater, excess of unlabeled E,, For the time course of [3H]E2 exchange, the hER-LBD was diluted more extensively so as to ensure that the E, solution would be saturating. It was diluted with 0.05 M Tris, 0.15 M NaCI, 20% glycerol, 1 mM DTT, and 0.1 mM EDTA, pH 7.4, at 25 C to give 10 nM ER. This resulted in 20 nM low specific activity E, present in solution as carry-over from the chromatographic purification. BSA was added as a carrier protein (2 mg/ml), and reversible binding was measured using the HAP assay (37). To label with TAZ, samples were incubated 0.5 h at 25 C. For exchange with E, or the photoaffinity ligand, hexestrol diazirine, incubations were conducted for 2 h at 25 C. After exchange with hexestrol diazirine, covalent attachment was effected by 15 min of photolysis at greater than 315 nm (41). Covalent attachment for either affinity ligand was assayed by the EtOH-disc assay (56). Labeling of the low specific activity PH]E, that is present with high specific activity ligands, the hER-LBD diluted 1 :lO with 50 mM Tris buffer (pH 7.4 at 25 Liquid Chromatography the method of Bearden using BSA. (39). (HPLC) HPLC analyses were performed on four reversed-phase columns using two systems. HPLC separations of the original protein preparation were performed with either a Vydac 2.1 mm x 250 mm C-4 reversed-phase column (5 pm particles, 300 A pore size) or a Vydac 2.1 mm x 250 mm C-6 reversedphase column (5 pm particles, 300 A pore size) using a SpectraSYSTEM (ThermoSeparations Inc., Carlsbad, California) HPLC system consisting of a model AS3000 autosample injector, a model P4000 gradient (piston) pump, and a model UV2000 dual wavelength UV detector outfitted with a standard analytical (9 ~1, 6 mm path) flow cell. UV detection was followed at 216 nm and 280 nm to observe amide and aromatic absorbances, respectively. This HPLC system was additionally outfitted with an P-RAM model 2 (IN/US Systems Inc., Tampa, FL) continuous-flow scintillation counter for the on-line detection of 3H-labeled receptor ligands. The HPLC interfaced with the ESI-MS probe used microbore RP-HPLC columns from Brownlee-Applied BioSystems MOL 656 END0 1995 Vol 9 No. 6 (1 .O mm id C-l 8, 7 pm particles, 300 A pore size), either 100 mm or 250 mm long; the longer column permitted loading of more injected material, while maintaining the low 40 yllmin eluent flow rate. This microbore system consisted of an Applied BioSvstems (Foster Citv. CA) model 140b dual svrinae pump equipped with two 101ml syringes and very low dead volume mixing tee, and an Applied BioSystems model 759a single wavelength UV detector with 2.5~~1 flow-through detection cell. Detection was set for the UV amide absorbance between 21 O-21 6 nm. Binary gradient elution conditions were used for all analyses. Compound linear gradients were generated for elution from 2% CH,CN to 100% CH,CN over 60-180 min; binary components for these gradients were comprised of solvent A (H,O, 0.06”/, CF,COOH) and solvent B (CH,CN, 0.05”/, CF,COOH). Solvents and reagents used for the HPLC and ESI-MS analyses were typically HPLC grade and were used without further purification. Water used was Milli-Q grade distilled before use. Acetonitrile was Optima (HPLC) grade as supplied by Fisher (Pittsburgh, PA). Trifluoroacetic acid (CH,COOH, TFA), protein sequencing grade (99% pure), was obtained from Aldrich Chemical Co (Milwaukee, WI) and used as supplied. ESI-MS A VG Quattro (quadrupole-hexapole-quadrupole, QHQ) mass spectrometer system (Fisons Instruments, VG Analytical; Manchester, UK) was used for the analysis of the receptor protein. Data aquisition and processing were controlled by the VG MassLynx (version 2.0) data system; MAXEnt (Maximum Entropy) software was used for the processing and analysis of zero charge state electrospray data for the receptor and large protein fragments. Collection of all electrospray data was performed on samples coupled through an HPLC/ ESI-MS interface with an Applied BioSystems microbore HPLC (see above) operating at 40 pl/min. Postcolumn eluent was split using static pressure balancing through a low dead volume mixing tee and a variable length of 75 pm id fused silica tubing. Empirically determined split ratios of 1:6 to 1:8 were established such that -5 pl/min HPLC eluent was directed to the ESI-MS probe for nebulization. This postcolumn splitting resulted in ESI-MS analysis of lo-30% of the injected sample and permitted the collection of 70-90% of desalted and purified protein fragments. HPLC sample injections of 5 ~1 were comprised typically of 15-30 pmol/pI receptor in buffer solution (2-5 M urea). The minimum quantity of receptor required for adequate detection under these conditions appears to be approximately lo-20 pmol at the ESI probe in a 5 $/min eluent flow. Cesium clusters [(Csl),Cs+], generated from freshly prepared solutions of cesium iodide (3 mg/ml) in .50:50 H,O: CH,CN, were used for external calibration of the ESI-MS from m/z 300-2300. Typical ESI data aquisition was performed for the protein using 4.9-set scans on 5.0-set intervals; the resulting total ion chromatograms (TIC) cover m/z 300-2300 (408.2 U/set). MALDI/Time of Flight (TOF)-MS A VG TOFSPEC matrix-assisted laser desorption/TOF mass spectrometer (Fisons Instruments, VG Analytical; Manchester, UK) using a 322 nm N, laser and 22 kV acceleration voltage was used for the analysis of the hER-LBD. BSA was used as an external calibration standard for all proteins assayed. [2-(4-Hydroxyphenylazo)benzoic acid] (HABA, Aldrich Chemical Co.) was found to be an effective matrix for the analysis of these protein solutions containing 2-5 M urea, provided a modification was made to the matrix deposition procedure (34): A saturated solution of HABA in 50:50 EtOH: CH,CN was spotted and dried on the target support; this matrix was overlayed with the hER-LBD sample, diluted lo- to 30-fold with 50:50 H,O-CH,CN, by the spotting of 2-3 ~1 of a l-3 pmol/$ solution of protein. After having been dried, the target spot was rinsed two to three times with 5 ~1 H,O to remove excess urea and other buffer salts and then allowed to redry. Samples thus prepared were analyzed by averaging 20-25 laser pulses. Observed masses of proteins prepared using this overlay/rinse procedure were generally 100300 Da higher than expected based on the BSA calibration and the masses observed by ESI-MS analysis. Acknowledgments We are grateful to Dr. Richard Milberg MALDI-MS and ESI-MS and to Drs. C. G. for helpful discussions. Dr. Greene gratefully technical assistance of Chris Hospelhorn Terminal analysis was performed by the Facility at the University of Illinois (Urbana, for assistance with Miller and K.-L. Ngai acknowledges the and Lin Cheng. NGenetic Engineering Illinois). Received December 13, 1994. Revision received February 21, 1995. Accepted March 13, 1995. Address requests for reprints to: Dr. John A. Katzenellenbogen, 461 Roger Adams Laboratory 37-5, Department of Chemistry, University of Illinois, 600 South Mathews Avenue, Urbana, Illinois 61801. This work was supported in part by grants from the NIH (PHS 5R37DK-15556 to J. A. K., and PHS 5ROl CA-02897 to G. L. G), the U.S. Army Medical Research Core (DAMD 17-94-J-44228 to G. L. G.), and the American Cancer Society (BEGIE to P. J. K.). 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Anal Biochem 166:368-379 Workshop on Heterogeneity of Calcium Microdomains and Cell Activation Villa Olmo-Como, Italy, November 34, 1995 Sponsored by The Calcium Club-European Chapter, Institute of Pharmacological Sciences, Milan Fondazione Giovanni Lorenzini, Milan and Houston. Program and lectures are as follows. November 3, 1995-Friday Afternoon: 1400-I 800: T. Meyer (Durham, USA) “Source of nuclear calcium signals,” R. Rizzuto (Padua, I) “Measuring organelle calcium with recombinant aequorin,” A. Konnerth (Homburg, D) “Dynamics and localization of calcium microdomains during synaptic activity,” and W. Mason (Cambridge, UK) “Real time confocal and video imaging of spatial and temporal heterogeneity in intracellular and intranuclear calcium signaling.” November 4, 1995-Saturday Morning: 0900-1230: A. Marty (Goettingen, D) “Calciuminduced calcium release in cerebellar Purkinje cells,” K.-H. Krause (Geneve, CH) “Ca*+ microdomains and site of lysosomal exocytosis in neutrophils,” Speaker to be announced, and Selected Oral Communications. The schedule for Saturday Afternoon (1330-1830) is as follows. Poster Session: F. Grohovaz (Milan, I) “Intracellular calcium mapping by analytical electron microscopy” and S. Bolsover (London, UK) “Nuclear calcium dynamics in nerve cells.” Round Table chaired by Prof. E. Carafoli: “Independent regulation of Ca*+ in the nucleus: yes or no?” Invited discussants: M. Mazzanti, T. Meyer, R. Rizzuto, L. Santella (plus selected brief communications). Send requests for information regarding Program and Registration Form to Organizing Secretariat: Daniela Galli, The Calcium Club, Institute of Pharmacological SciencesUniversity of Milan, Via G. Balzaretti g-20133 Milan (Italy), Phones: (+ 39/2) 20.488.320, 20.488.312, 20.488.306. Fax: (+39/2) 2940.4961. Information regarding scientific aspects of the program may be requested from Scientific Secretary: Simonetta Nicosia.