ISSN 10681620, Russian Journal of Bioorganic Chemistry, 2010, Vol. 36, No. 5, pp. 603–609. © Pleiades Publishing, Ltd., 2010. Original Russian Text © N.F. Khabibullina, E.N. Lyukmanova, G.S. Kopeina, Z.O. Shenkarev, A.S. Arsen’ev, D.A. Dolgikh, M.P. Kirpichnikov, 2010, published in Bioorgan icheskaya Khimiya, 2010, Vol. 36, No. 5, pp. 654–660.
Development and Optimization of a Coupled CellFree System for the Synthesis of the Transmembrane Domain of the Receptor Tyrosine Kinase ErbB3 N. F. Khabibullinaa, b, E. N. Lyukmanovab,1, G. S. Kopeinab, Z. O. Shenkarevb, A. S. Arsen’evb, D. A. Dolgikha, b, 1, and M. P. Kirpichnikova, b a
b
Moscow State University, Moscow, 119991 Russia Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. MiklukhoMaklaya 16/10, Moscow, 117997 Russia Received February 9, 2010; in final form, February 15, 2010
Abstract—A coupled cellfree expression system (CECF) for the production of the transmembrane domain of the human receptor tyrosine kinase ErbB3 (residues from 632 to 675) has been developed based on the Escherichia coli S30 extract. The synthesis of the domain in the soluble form in the presence of various deter gents and in the form of an insoluble precipitate of the reaction mixture has been examined. The conditions for the purification of the recombinant domain obtained using the two approaches have been determined. The final yield of the target protein under optimal conditions was 1.8–2.0 mg per 1 ml of the reaction mixture. Key words: coupled cellfree expression system, membrane proteins, tyrosine kinases DOI: 10.1134/S1068162010050080 21
INTRODUCTION
According to the presentday prognoses, about half of all human membrane proteins are potential targets for the development of novel drugs [1–3].2 Despite the wide diversity and great practical importance of MPs, the progress in their studies has been hindered since their native structure is retained only in the presence of a biological membrane or an appropriate membrane mimet medium [4, 5]. In recent years, the use of CECFs for the production of recombinant proteins has become increasingly popular [6–10]. The yields of the target proteins in these systems currently reach several milligrams per one milliliter of RM, which enables one to obtain protein preparations in amounts necessary for biological, biochemical, and biophysical investigations. CECFs have some advantages over sys tems based on cell production [4]. The major advan tage is that it is possible to add cofactors, inhibitors, and ligands to RM that promote the synthesis of the 1 Corresponding authors; phone: (495) 3352888; fax: (495) 330
6983; email: ekaterina
[email protected] and
[email protected]. 2 Abbreviations: Brij35, dodecylpolyoxyethylene35; Brij58, hexadecylpolyoxyethylene58; Brij78, octadecylpolyoxyethyl ene78; Brij98, octadecylpolyoxyethylene98; CECF, coupled cellfree expression system; MP, membrane protein; FM, feed ing mixture; SDS, sodium dodecyl sulfate; Triton X100, 4(1,1,3,3teteramethylbutyl)phenyl polyethylene glycol; TMD, the transmembrane domain; TMErbB3, the transmem brane domain of the receptor tyrosine kinase ErbB3; RM, reac tion mixture.
protein in the native conformation. For the produc tion of MPs, components maintaining MPs in the sol uble form, such as detergent micelles, liposomes, or lipoprotein particles, are added directly to RM [2, 4, 5, 11–14]. In addition, using CECFs it is easy to obtain proteins selectively labeled at particular amino acids residues by stable or radioactive isotopes [9, 15]. The family of receptors of the human epidermal growth factor consists of four transmembrane tyrosine kinases: ErbB1, ErbB2, ErbB3, and ErbB4 [16]. These proteins contain a variable extracellular Nterminal domain responsible for the binding of ligands; a mem brane domain formed by one transmembrane helix; and the cytoplasmic region, which involves a per imembrane domain, a catalytic tyrosine kinase domain, and a Cterminal regulatory domain [17]. Owing to the interactions of tyrosine kinase and trans membrane domains, the receptors of the ErbB family form catalytically inactive homo or heterodimeric complexes in the cell membrane. During ligand bind ing, these receptor dimers undergo conformational rearrangements, which lead to the activation of one of the intracellular tyrosine kinase domains [18–20]. Because receptor tyrosine kinases affect cell growth and development, their dysfunction is often the cause of malignant cell transformation [17]. The intracellu lar tyrosine kinase domain of the receptor of ErbB3 has no intrinsic enzymatic activity. Nevertheless, because the receptors of this type can function in the form of heterodimers with other members of the ErbB
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Fig. 1. Schematic diagram of the arrangement of a CECF of the dialysis type (a) and the vector pET22b(+)/TMErbB3 (b).
family [20], the structural and functional studies of TMErbB3 are of interest from the viewpoint of creat ing novel medicinal preparations for the controlled regulation of the functioning of the epidermal growth factor receptors. TMDs of receptor tyrosine kinases were earlier obtained by chemical synthesis [17, 21] and the use of bacterial expression systems [22]. In bacterial systems, the TMD of the receptor tyrosine kinase ErbB2 was produced as thioredoxinfused constructs [22]. The isolation and purification of TMDs were carried out in several steps: the purification of a hybrid protein was fol lowed by its enzymatic hydrolysis, after which the TMD itself was purified [22]. The synthesis of TMErbB3 in bacterial cells, as distinct from the production of TMDs of other tyrosine kinases, was ineffective, and the yield of the target protein was insufficient to per form structural and functional studies of this domain. In the present study, TMErbB3 was produced using an alternative approach, which involves a CECF based on the E. coli S30 extract. In this system, an indi vidual TMErbB3 was produced without using special partner proteins. It was shown that the addition of detergents (Brij35, Brij58, Brij78, Brij98, and Tri ton X100) into RM promotes the production of TMD in the soluble form. We compared the efficiency of TMErbB3 production in the absence and presence of various detergents and developed protocols for the iso lation and onestep purification of the target product under optimal conditions. RESULTS AND DISCUSSION Optimization of CECF of TMErbB. A CECF of the dialysis type based on the E. coli S30 extract was used in which FM and RM were separated by a semiperme able membrane (Fig. 1a) [23]. The pET22b(+)/TM ErbB3 plasmid vector containing the sequence encod ing human TMErbB3 (residues from 632 to 675; MQTLVLIGKTHLTMALTVIAGLVVIFMMLG GTFLYWRGRRIQNKRHHHHHH) with an addi
tional six His residues at the Cterminus for the subse quent purification of the target protein by metal affin ity chromatography served as a template (Fig. 1b). The use of the CECF requires the search of the optimal conditions for each newly obtained extract. Usually the concentrations of magnesium and potas sium ions, as well as the vector containing the gene of the target protein, are optimized [24]. In the present study, we performed the stepbystep optimization of TMErbB3 synthesis. First, the concentration of mag nesium acetate in the range from 9 to 14 mM at fixed concentrations of potassium (110 mM) and the vector (0.2 mg/ml) was optimized, and then the potassium acetate concentration in the range from 70 to 120 mM at the found optimal magnesium concentration and a vector concentration of 0.2 mg/ml was optimized. The concentration of vector pET22b(+)/TMErbB3 was optimized in the range from 0.1 to 0.5 mg/ml under the conditions found for magnesium and potassium. An analysis of the efficiency of TMErbB synthesis indicated that for the S30 extract, the optimal concen trations of magnesium and potassium ions and the vector are 11 mM, 80 mM, and 0.3 mg/ml, respec tively (Figs. 2a–2c). Synthesis of TMErbB3 in the form of a RM precipi tate. Membrane proteins can be obtained in a CECF either in the form of a RM precipitate or in the soluble form in the presence of a membranemodulating medium, e.g., detergent micelles. At the first stage, TMErbB3 was synthesized in the absence of deter gents. It was shown that in this case, the protein accu mulates as a waterinsoluble precipitate of RM (table). The TMD was isolated and purified by solubilization the RM precipitate in SDS with the subsequent metal affinity chromatography in the presence of SDS (Fig. 3, lanes 1–5). The resulting TMErbB3 prepara tion had a high degree of purity (>95%) and contained no highmolecularweight aggregates (Fig. 4b). The yield of the target product was ~2.0 mg/ml. A mass spectrometric analysis of the preparation showed that the mass of the protein synthesized (5972.2 Da) corre
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Fig. 2. Analysis of the synthesis efficiency of the TMErbB3 total amount depending on the concentration of (a) magnesium ions, (b) potassium ions, and (c) vector pET22b(+)/TMErbB3 and an analysis of the efficiency of the synthesis of soluble TMErbB3 depending on the concentration of detergents Brij35, Brij58, Brij78, Brij98, and Triton X100 (d). The level of soluble TMErbB3 synthesized in the absence of detergents is designated by the symbol “C”. Each value represents the average of two to three experiments; the systematic error is no greater than 16%. The content of the TMErbB3 preparation was estimated in com parison with the amount of purified TMErbB3 synthesized in the presence of 1% Brij58.
sponds to the calculated value. A CD spectroscopy analysis of a TMErbB3 preparation solubilized in SDS micelles showed that the content of helical ele ments in the secondary protein structure is about 50% (Fig. 4a), which agrees well with the data obtained previously for other TMDs of receptor tyrosine kinases of the ErbB family (~60%). An insignificant decrease in the percentage of the helical structure in TMErbB3 is probably related to the presence of an additional methionine residue at the Nterminus and six histidine residues at the Cterminus of the protein molecule. Synthesis of TMErbB3 in the soluble form in the presence of detergent micelles. CECF is an open sys tem, which makes it possible to add to the RM various substances and agents that increase the solubility of the molecules synthesized [9]. Among them are, in particular, nonionic detergents, which create an artifi cial membranemimet environment necessary for the stabilization of the MP structure in the solution [5, 9]. However, not all detergents are suitable for use in CECF [8]. Thus, dodecylphosphocholine (a detergent widely used in structural studies of MPs [25–27])
often inhibits the functioning of the CECF, which sig nificantly decreases the efficiency of MP synthesis [8]. Therefore, the production of MPs in cellfree expres sion systems requires a careful choice of detergents. Several nonionic detergents (Brij35, Brij58, Brij78, Brij98, and Triton X100) were tried in the synthesis of soluble TMErbB3. An analysis of the effectiveness of protein synthesis showed that the addi tion of detergents substantially increases the portion of the TMD present in the soluble fraction of RM (table, Fig. 2d). It was shown for Brij35, Brij58, Brij78, and Brij98 that increasing the detergent concentra tion from 1 to 2% did not lead to any significant increase in the content of soluble TMErbB3 (Fig. 2d). The yield of the soluble domain was the highest with the use of Brij58 and Brij78; the share of the soluble protein was also maximal with these deter gents (table). Triton X100 increased the TMErbB3 solubility (table); however, it markedly decreased the total yield of the product. The yield of the target prod uct monotonically decreased as the Triton X100 con centration increased from 0.2 to 2% (Fig. 2d).
Solubility of TMErbB3 synthesized in the absence and presence of different detergents Detergent, 1% Percentage of soluble TMErbB3 of the total amount of the domain synthesized, %
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Fig. 3. Analysis of the purification of TMErbB3 from a RM precipitate in the presence of 1% SDS (1⎯5) and of soluble TMErbB3 synthesized in the presence of Brij58 either without (6⎯10) or with Brij58 exchange for SDS (11⎯15): breakthrough (1, 6, 11); washing by buffer A (2, 7, 12); elution by 100 mM (3, 8, 13), 300 mM (4, 9, 14), and 500 mM imidazole (5, 10, 15); and 16, molecular mass marker. The TMErbB3 preparation is shown by an arrow.
The possibility of purifying the soluble TMErbB3 was examined using a preparation obtained in the presence of Brij58. The purification was carried out by metal affinity chromatography using two methods: in the presence of Brij58 and by exchange of this detergent or SDS during chromatography (Fig. 3, lanes 6–10 and 11–15, respectively). Despite the dif ferent purification protocols, the resulting prepara tions of soluble TMErbB3 exhibited similar physical chemical properties, were of a high purity grade, and contained no highmolecularweight aggregates. The yield of the TMD in both cases was ~ 1.8 mg/ml of RM. The CD spectra of TMErbB3 in the presence of Brij58 and SDS micelles were similar to that of the domain obtained from the RM precipitate (data not shown). These data indicate that the secondary struc ture of TMErbB3 does not depend on the method of synthesis. The successful application of Brij detergents for obtaining soluble TMErbB3 agrees well with the results of other studies. Thus, these detergents have earlier been used for producing some G proteincou pled receptors in CECF [4, 8, 28]. These studies have demonstrated the successful synthesis of porcine and human typeII vasopressin receptors and a human B type endothelin receptor with a yield of 6 mg/ml of RM for the porcine receptor and 3 mg/ml of RM for the human receptors in the presence of Brij58 and Brij78 at concentrations of 2 and 1%, respectively. In the case of the b subunit of E. coli F1F0 ATP synthetase, the highest yield of the protein in the soluble form (0.6 mg/ml) was also obtained in the presence of Brij 58 and Brij78 [29]. Triton X100 and digitonin dem onstrated a considerably weaker effect on protein sol ubility, and the application of Tween20 did not con tribute to the solubility of the b subunit of E. coli F1F0 ATP synthetase at all [29]. It should be noted that Tri
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Wavelength, nm Fig. 4. CD spectrum (a) and electrophoretic analysis (b) of the TMErbB3 preparation obtained from a RM precipi tate and purified by metal affinity chromatography (1, molecular mass marker; and 2, TMErbB3 preparation).
ton X100 was earlier used for the synthesis of the membrane protein Tsx, a transporter of nucleosides in the E. coli outer membrane [8]. By adding 0.1% Triton X100, it was possible to obtain soluble Tsx with a yield of 1 mg/ml of RM [8]. However, further increasing the detergent concentration led to a decrease in the level of the protein. These results agree well with the data obtained in the present study. Thus, we developed a highly effective system for the cellfree synthesis of the TMD of the human receptor tyrosine kinase ErbB3. Different approaches to the production of the protein were tried. We showed that TMErbB3 is produced with an approximately equal efficiency both as a RM precipitate and in the soluble form in the presence of several detergents. The elec trophoresis, mass spectrometry, and CD spectroscopy of purified TMErbB3 preparations obtained by dif ferent methods did not reveal any substantial differ ences in the physicalchemical properties of the pro tein. This indicates that both approaches can be used with equal effectiveness for the production of TMDs of membrane proteins. The developed system opens up new possibilities for structural and functional stud ies of TMErbB3. EXPERIMENTAL Preparation of a plasmid for the cellfree synthesis of TMErbB3. The gene encoding TMErbB3 was derived from the previously developed construct for expression in E. coli cells by PCR using the follow ing oligonucleotides (5'3'): CGCGCATATGCA GACCCTGGTTCTGAT and GCGGGATCCTTA CTAGTGATGGTGGTGATGGTGACGTTTGTT.
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Then, the gene encoding TMErbB3 was cloned into the commercial vector pET22b(+) (Novagen, United States) at the NdeI and BamHI restriction endonuclease sites. The resulting vector was named pET22b(+)/ТМErbB3. Production of plasmid pET22b(+)/ТМErbB3 in preparative amounts. For further study, the pET22b(+)/ТМErbB3 plasmid was produced in 0.6–0.8 l of LB nutrient medium containing ampicil lin at a concentration of 100 µg/l. The isolation and purification of the plasmid were performed using a Plasmid Maxi Kit (Qiagene, Germany). After the iso lation, the phenol deproteinization of the plasmid was carried out according to the manufacturers’ protocol by adding 1 volume of mixture phenol–chloroform– isoamyl alcohol (25 : 24 : 1) followed by centrifugation at 14000 rpm for 10 min. The aqueous phase was with drawn, treated with an equal volume of chloroform, and centrifuged as described above. The aqueous phase was withdrawn again; 1/10 of the volume of 3 M NaOAc, pH 5.0 (Sigma, United States), and 2.5 vol umes of 96% ethanol were added; and the mixture was left to incubate for 1 h at –70°C. After centrifuging the mixture at 14000 rpm for 20 min at +4°C, the sedi ment was washed twice with 70% ethanol and then with 96% ethanol, airdried, and dissolved in water. The concentration of plasmid DNA was determined from absorption at 260 nm. Production of TMErbB3 in CECF. The S30 extract from E. coli (strain A19) was obtained using the protocol developed in [30, 31]. The synthesis in a cell free system based on the E. coli S30 extract was per formed according to the protocols reported in [31]. T7 polymerase was obtained using the protocol described in [32]. The enzyme obtained was compared accord ing to activity with a commercial T7 polymerase prep aration (Fermentas, Lithuania). The final concentra tions of the RM components were as follows: 100 mM HEPESKOH (Fluka, United States), pH 8.0; 11 mM Mg(OAc)2, 80 mM KOAc, 20 mM potassium acetyl phosphate (Sigma, United States); 20 mM potassium phosphoenolpyruvate (Aldrich, United States); a set of amino acids (1.3 mM each) except for Arg, Cys, Met, Trp, Asp, and Glu, the concentration of each amino acid being 2.3 mM [8]; 0.15 mg/ml of folic acid (Sigma); each of the 4ribonucleoside triphosphates (1 mM), XI Complete protease inhibitor® (Roche Diagnostics, Germany); 0.05% NaN3, 2% polyethyl ene glycol 8000 (Sigma); 0.3 unit/µl of RiboLock ribonuclease inhibitor (Fermentas, Lithuania); 0.04 mg/ml of pyruvate kinase (Fermentas, Lithua nia); 5.5 µg/ml of T7 polymerase, 0.3 mg/ml of plas mid DNA, 0.5 mg/ml of total tRNA (from E. coli MRE 600) (Roche Diagnostics, Switzerland); and an E. coli S30 extract (30% of the total volume of the mix ture). For the synthesis of TMErbB3 in the soluble form, detergents Brij35, Brij58, Brij78, Brij98, and Tri ton X100 (Sigma, United States) were added to RM RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
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to a final weight concentration of 1%. FM had the same composition as RM, excluding the highmolec ularweight components, such as the S30 extract, plas mid, enzymes, and ribonuclease inhibitor. The RM was placed in a dialysis bag of a volume of 1.5 ml from the semipermeable cellulose membrane (pore size 12 kDa; Sigma, United States), which was immersed in FM. The RM–FM ratio was 1 : 15. TMErbB3 was synthesized in test vials of a volume of 50 ml using an HBMinidizer incubator (UVP, United States) at a velocity of vials rotation of 12 rpm. The incubation was carried out for 20–22 h at 30°C. After the synthesis, the RM containing TMErbB3 was taken out from the dialysis bag and exposed to further procedures. Analysis of the efficiency of TMErbB3 synthesis. Proteins were separated by 12% PAGE in a Tristricine buffer system supplemented with 0.1% SDS and trans ferred to a TransBlot® Transfer Medium nitrocellu lose membrane (BioRad, United States) using a SemiDry Electroblotter (BioTecMed ApS, Den mark). A Prestained Protein Molecular Weight marker (19.5–118 kDa) (Fermentas, Lithuania) was used as a molecular mass marker. Membranes were incubated with mouse monoclonal antibodies (Histag® Mono clonal antibody; Novagen, United States) obtained against the hexahistidine sequence and then with goat antimouse IgG alkaline phosphatase (Novagen, United States) conjugated with horseradish peroxi dase. Protein bands were developed by keeping a membrane for 5 min in a solution of substrates BCIP/NBT (5bromo4chloro3indolylphos phate/nitroblue tetrazolium). After the development, the membrane was washed with distilled water. The developed membranes were scanned on a plotting board scanner, and the images were processed using the OptiQuant program version 3.00 (Packard Instru ment Company, United States). Isolation and purification of TMErbB3. If the pro tein was obtained as a precipitate, the RM after the synthesis was centrifuged for 15 min at 14000 rpm. The sediment was dissolved in buffer A (20 mM Tris HCl, 250 mM NaCl, 1 mM NaN3, pH 8.0) supple mented with 1% SDS, with 3 ml of the buffer per 1 mg of protein. Then the solution was clarified by centrifu gation for 10 min at 14000 rpm. The NiSepharose Fast Flow sorbent (GE Healthcare, Sweden) was equilibrated in buffer A in the presence of 1% SDS. A protein sample was incubated with the sorbent for 1 h at room temperature, after which the resin with the immobilized protein preparation was transferred to a PM10 column (GE Healthcare, Sweden). The col umn was washed with ten volumes of buffer A contain ing 1% SDS with the addition of 10 mM imidazole. The protein was eluted stepwise by buffer A containing 1% SDS and imidazole at a concentration of 100, 300, and 500 mM. If the protein was obtained in the soluble form in the presence of Brij58, the RM was clarified by cen trifugation for 10 min at 14000 rpm. Then the super Vol. 36
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natant was incubated for 1 h at room temperature with a NiSepharose Fast Flow resin equilibrated in buffer A supplemented with either 1% Brij58 or 1% SDS. Further purification of TMErbB3 was carried out as in the case of the protein obtained as a precipitate by performing all manipulations in the presence of the appropriate detergent. The concentration of the pro tein in fractions obtained by chromatography was determined spectrophotometrically from absorption at 280 nm using the molar absorption coefficient (ε280 6990). Mass spectrometry analysis of TMErbB3. In the mass spectrometry analysis of a TMErbB3 prepara tion obtained as a RM precipitate, a precipitate was dissolved in 70% formic acid. The TMErbB3 prepa ration obtained in the presence of Brij58 was purified by metal affinity chromatography and reprecipitated by 10% trichloroacetic acid. The precipitate was dis solved in 70% formic acid. The mass spectra of both preparations were obtained on an UltraFlex TOF/TOF timeofflight mass spectrometer (Bruker Daltonics, Germany) equipped with a MALDI ion ization source. Positively charged ions were detected in a linear regime. CD spectroscopy analysis of the secondary struc ture of TMErbB3. CD spectra were recorded at room temperature on a J810 spectrometer (Jasco, Japan). In the samples, the concentration of the proteins examined was 0.05 mM. The buffer contained 20 mM TrisHCl, 250 mM NaCl, and 1% of detergent, pH 8.0. The content of the secondary structure ele ments was determined by the Dichro Web program using the Contin LL algorithm.
3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
14.
ACKNOWLEDGMENTS The authors are grateful to Ya.S. Ermolyuk for the vector containing the gene of TMErbB3 and to O.V. Vorontsova for her help in experiments by CDspectroscopy method. This work was performed within the framework of the Federal Target Program “Scientific and Pedagogi cal Specialists of Innovation Russia” (2009–2013) and was partially supported by the Russian Academy of Sciences (program “Cellular and Molecular Biol ogy”), Russian Foundation for Basic Research, and the grant from the President of the Russian Federation (MK8404.2010.4).
15. 16. 17. 18. 19.
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