Purification and in Vitro Folding of Recombinant Human Thrombopoietin Receptor Expressed in Escherichia coli

Protein Expression and Purification 21, 129 –133 (2001) doi:10.1006/prep.2000.1341, available online at http://www.idealibrary.com on

Purification and in Vitro Folding of Recombinant Human Thrombopoietin Receptor Expressed in Escherichia coli Christine K. F. Ng, Susan Huxtable, Pelin Xu, and Dennis P. H. Hsieh 1 Department of Biology, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China

Received June 27, 2000, and in revised form August 30, 2000

Thrombopoietin receptor (TPOR) is a member of the cytokine receptor superfamily expressed primarily on hematopoietic cells. TPOR plays an important role in regulating platelet production. Due to its low expression level in human tissue, studies on the biochemical and biophysical properties of TPOR have been limited. In the present study, an extracellular domain of recombinant human TPOR (rh TPOR-EN) was expressed in Escherichia coli as inclusion bodies. Purification was achieved by metal chelated chromatography under denaturing condition and was refolded by gel filtration chromatography. Far UV circular dichroism spectroscopy and surface plasmon resonance experiments were performed to demonstrate that the protein was in a refolded state and could bind with its ligand. Thus, a production and purification scheme was developed by which sufficient quantities of rh TPOR-EN can be made available for biochemical and biophysical characterizations. © 2001 Academic Press

Thrombopoietin receptor gene (c-mpl) 2 is a cellular homolog of the murine myeloproliferative leukemia virus (MPLV) envelope gene, v-mpl (1). TPOR is a member of the hematopoietic receptor superfamily and is primarily expressed in placenta, bone marrow, fetal liver (but not adult liver), fetal blood (34 weeks), cord blood, peripheral blood, and phytohemagglutinin-stim1 To whom correspondence and reprint requests should be addressed. Fax: (852) 2358-1559. E-mail: [email protected]. 2 Abbreviations used: c-mpl, thrombopoietin gene; MPLV, myeloproliferative leukemia virus; v-mpl, MPLV envelope gene; TPOR, thrombopoietin receptor; rh TPOR-EN, recombinant human TPOR; LB, Luria–Bertani; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; DTT, dithiothreitol; CD, circular dichorism; SPR, surface plasmon resonance; NHS/EDC, N-hydroxysuccinimide/N-ethyl-N⬘-(dimethylamino-propyl)carbodiimide; RU, resonance unit; BSA, bovine serum albumin; IPTG, isopropyl thio-␤-D-galactoside.

1046-5928/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

ulated lymphocytes (2). Amino acid sequences of TPOR indicates that the rh TPOR-EN contains a 200-aminoacid motif that includes two domains, each composed of seven ␤ strands. Of the two domains, the first displays distinctive conservation of four cysteine residues at its amino terminus and a Trp-Ser-X-Trp-Ser or WSXWS motif near the carboxyl terminus (3). It has been experimentally demonstrated that the TPOR-EN is the site of binding with the ligand TPO (4). Functional studies have shown that exposure of CD34⫹ cells to TPOR antisense oligodeoxynucleotides selectively inhibited megakaryocytic colony formation in vitro without affecting the formation of erythroid and granulocytic-macrophage colonies (5). In addition, an 85% reduction in peripheral platelet number was observed in homozygous TPOR gene knock-out mice while leaving other hematopoietic lineages unaffected. Together, these observations indicate that TPOR plays an important role in megakaryocytopoiesis and platelet production. The discovery of TPOR has stimulated intense interest in the study of megakaryocytopoiesis and platelet production. However, hindered by low expression level of TPOR in vivo, detailed study of the biochemical and biophysical characteristics of TPOR has not been possible. So far as we know, human TPOR has not previously been expressed in large quantities in any bacterial system. In the present study, we set out to work on the overexpression of rh TPOR-EN in an E. coli system. The product, upon purification and in vitro folding, was demonstrated to be biochemically active in specifically binding with its ligand TPO. Thus, the bacterial expression system and the purification scheme developed in this study were shown to be useful in producing milligram quantities of rh TPOR-EN for biochemical and biophysical characterizations. 129

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MATERIALS AND METHODS

Materials Fetal liver mRNA was purchased from Clontech (CA). All primers used for RT-PCR and SuperScript II RNase H-reverse transcriptase were purchased from Gibco-BRL Life Technologies (Gaithersburg, MD). A GeneAmp PCR reagent kit was purchased from Perkin Elmer (CT). All restriction enzymes, FPLC System, HiTrap affinity column, and Superdex 75 HR 10/30 gel filtration column were purchased from Amersham Pharmacia Biotech. (Uppsala, Sweden). The pET-30a expression vector, BL21(DE3)pLysS (F- ompT hsdSB(rB-mB-) gal dcm (DE3) pLysS), was purchased from Novagen (Madison, WI). A Jasco Model J-720 spectropolarimeter was purchased from Japan Spectroscopic (Tokyo, Japan). An IAsys affinity biosensor, a NHS coupling kit, and a CM-Dextran curvette were purchased from Affinity Sensor (Paramus, NJ). rh TPO was purchased from Pepro Tech (London, UK). Other chemicals not specified were obtained from Sigma (St. Louis, MO). Cloning and Expression of rh TPOR-EN The rh TPOR-EN was synthesized by two overlapping cDNAs (encoding domain 1 and domain 2, respectively) from the human fetal liver messenger RNA (Clontech) by RT-PCR. The oligonucleotide primers 5⬘CCG GAA TTC CAA GAT GTC TCC TTG CTG GCA TCA GA-3⬘ (containing a EcoRI restriction site, underlined) and 5⬘-GGC CGC TCG AGT TAA GTC ACA GGG AGG GAC CAG G-3⬘ (containing an XhoI restriction site, underlined) were used to amplify domain 1, and oligonucleotide primers 5⬘-CCG GAA TTC TGG CTG CAG CTG CGC AGC GAAC-3⬘ (containing a EcoRI restriction site, underlined) and 5⬘-GGC CGC TCG AGT TAC AGG CGG TCT CGG TGG CGG TCT-3⬘ (containing an XhoI restriction site, underlined) were used to amplify domain 2. The PCR products were digested with restriction endonucleases EcoRI, XhoI, and BamHI, purified, and were ligated to form the rh TPOR-EN. The ligated product was subcloned into the kanamycin resistant vector pET 30a. The construct was confirmed by nucleotide sequencing and was used to transform E. coli strain BL21(DE3)pLysS. Expression of rh TPOR-EN A 1-L culture of transformed E. coli was grown in aerated LB medium supplemented with 30 ␮g/mL of kanamycin at 37°C to a density of A 650 nm ⫽ 0.6. Expression of the rh TPOR-EN fusion protein was induced by adding isopropyl thio-␤-D-galactoside to a final concentration of 1 mM, and the culture was incubated at 30°C further for 3 h. The bacteria were harvested by centrifugation at 4420g for 15 min at 4°C and the pellet stored at ⫺20°C.

Isolation and Solubilization of rh TPOR-EN Inclusion Bodies The cell pellet was resuspended with 100 mL of 50 mM Tris–HCl, pH 8.0, with 2 mM EDTA and 10 mL of 1% Triton X-100. The mixture was chilled on ice for 30 min and was lysed by sonication. The lysate was centrifuged at 4420g for 15 min at 4°C, and the isolated inclusion bodies were further purified by repeated washing and differential centrifugation in a binding buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris–HCl, pH 7.9). The purified inclusion bodies were resuspended in 100 mL of 8 M urea freshly prepared in the binding buffer for 2 h at 4°C. The 2-mercaptoethanol was added to a final concentration of 10 mM, and the mixture was kept at 4°C for at least 1 h. Purification of rh TPOR-EN The solubilized inclusion bodies were centrifuged at 12,000g for 1 min and the supernatant was applied to the metal chelated column, Hi Trap affinity column, charged with 2.5 mL of 0.1 M CuSO 4, and preequilibrated with 8 M urea freshly prepared in the binding buffer. The column was washed with 20 mL of 8 M urea in the binding buffer. The bound protein was eluted with a step imidazole gradient which was a result of mixing the binding buffer with different percentage of the elution buffer (8 M urea, 1 M imidazole, 0.5 M NaCl, 20 mM Tris–HCl, pH 7.9) at a flow rate of 5 mL/min. Samples were collected in 1-mL fractions. The fractions containing the purified protein were analyzed by SDS–PAGE with Coomassie blue staining. Size-Exclusion Chromatography Fractions containing rh TPOR-EN were pooled and subjected to in vitro folding by passing through a gel filtration column, Superdex 75 HR 10/30 gel filtration column preequilibrated with 100 mL of a refolding buffer (20 mM NaHPO 4, 200 mM NaCl, 1 mM DTT, pH 7.4) at a flow rate of 0.5 mL/min. Fractions of 1 mL were collected and analyzed by SDS–PAGE with Coomassie blue staining and far UV CD spectroscopy. Far UV CD Spectroscopy Far UV CD spectroscopy was carried out on a Jasco Model J-720 spectropolarimeter. The instrument was calibrated with a standard, d-10-camphor sulfonate at 0.06% (w/v) in a 1-cm pathlength cuvette. rh TPOR-EN was prepared by dialysis in a buffer containing 5 mM imidazole, 0.5 M NaCl, and 20 mM Tris–HCl, pH 7.5, at a protein concentration of 0.25 mg/mL. Spectral information was recorded at 25°C from 190 to 250 nm. The following settings were used: bandwidth, 1 nm; time constant, 2.0 s; step resolution, 0.1 nm; scan speed, 10 millidegree/min; sensitivity, 1 millidegree/cm. The fi-

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FIG. 1. Construction of the rh TPOR-EN-pET 30a expression vector. rh TPOR-EN was cloned into pET 30a at the EcoRI and XhoI site. A polyhistidine tag was linked to the N-terminus and the resulting plasmid was designated as rh TPOR-EN-pET 30a.

nal spectrum was expressed as an average of five scans after subtraction of the buffer baseline. Surface Plasmon Resonance Binding Experiment The association and dissociation rate constants of the interaction between an immobilized rh TPO, and the rh TPOR-EN produced in this study were determined by surface plasmon resonance (SPR) measurements employing an IAsys instrument (Affinity Sensor, USA). The rh TPO was immobilized onto a CMDextran cuvette using the amine coupling method described in the IAsys manual. Briefly, the rh TPO was diluted to a final concentration of 0.1 mg/mL with 10 mM sodium acetate at pH 5.0. The sensor chip matrix was activated for 6 min with N-hydroxysuccinimide/Nethyl-N⬘-(dimethylamino-propyl)carbodiimide (NHS/ EDC) prior to injection of rh TPO in coupling buffer for 4 min. The unoccupied binding sites were blocked by

FIG. 2. SDS–PAGE gel (12%) of rh TPOR-EN expression and purification. Lane 1, crude extract of E. coli before induction; lane 2, crude extract of E. coli induced with 1 mM IPTG for 3 h at 30°C; lane 3, soluble protein fraction; lane 4, inclusion bodies solubilized in 8 M urea; lane 5, pooled fractions of rh TPOR-EN eluted from immobilized metal affinity chromatography; lane 6, pooled fractions of rh TPOR-EN refolded from gel filtration chromatography.

FIG. 3. SDS–PAGE gel (12%) showing protein lysate of E. coli prior to induction and following induction with different concentrations of IPTG and different induction periods at 30 and 37°C. u, prior to IPTG induction; a, 0.4 mM IPTG; b, 0.6 mM IPTG; c, 0.8 mM IPTG; d, 1 mM IPTG.

injecting 1 M ethanolamine for 5 min. After immobilization, the flow cell was washed with 20 mM HCl and PBST (10 mM sodium phosphate, 2.7 mM potassium chloride, 138 mM sodium chloride, and 0.05% (v/v)

FIG. 4. Metal affinity chromatography of rh TPOR-EN inclusion bodies solubilized in 1⫻ binding buffer with 8 M urea.

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FIG. 5. SDS–PAGE gel (12%) showing the purity of protein fractions corresponding to the metal affinity chromatography (Fig. 4).

Tween 20). Binding affinity of the immobilized rh TPO with rh TPOR-EN was determined by passing different concentrations of rh TPOR-EN in 10 mM sodium acetate, ranging from 16.9 to 169.5 nM, over the immobilized rh TPO cuvette for 5 min and was dissociated with 10 mM acetate buffer for 10 min. All binding experiments were conducted at 25°C. At the end of each binding cycle, the biosensor chip surface was regenerated by exposure to 20 mM HCl for 1 min. The SPR signal obtained in each binding cycle was recorded as a real time pattern plotted in resonance units, RU, against time. The association and dissociation constants were calculated by using the FASTfit program algorithm. RESULTS AND DISCUSSION

Construction of rh TPOR-EN was achieved by ligating two overlapping cDNA fragments to obtain a 1416-bp cDNA. The latter cDNA was inserted at the EcoRI site downstream of a T 7 promoter in pET 30a to give a construct, rh TPOR-EN-pET 30a (Fig. 1). DNA sequencing confirmed that the sequence was correct and was in frame with the histidine tag on the pET 30a. The construct was used to transform E. coli BL21(DE3)pLysS as a bacterial expression host, to carry out a high level expression of rh TPOR-EN. In the crude extract of E. coli, the strong band on the SDS–PAGE gel in the region close to the molecular mass of 59 kDa, which is close to the molecular mass calculated for the protein based on its amino acid sequence (Fig. 2). As shown by cell fractionation (Fig. 2), rh TPOR-EN was mainly present in the insoluble pro-

FIG. 6. Gel filtration chromatogram of purified rh TPOR-EN on Superdex 75 column.

tein fraction. The amount found in the soluble protein fraction was insignificant. Since inclusion body formation can sometimes be prevented by altering the growth conditions of the bacteria (6), a number of different growth conditions were tried. Expression of soluble rh TPOR-EN was not increased by incubation at a lower temperature (30°C), induction with different IPTG concentrations (0.4, 0.6, 0.8, 1 mM), or with different induction periods (1, 2, 3 h) (Fig. 3). When purified with immobilized metal affinity chromatography, three protein peaks were detected at 280 nm (Fig. 4). SDS–PAGE (Fig. 5) revealed that the first

TABLE 1 Purification of rh TPOR-EN from 1 L of E. coli BL21 (DE3) pLysS

Fraction

Total protein (mg)

Yield (%)

Inclusion bodies His Trap affinity column eluate Superdex 75 gel filtration column eluate

72.5 40.9 3.16

100 56.4 4.4

FIG. 7. SDS–PAGE gel (12%) showing the proteins in fractions 4 and 5 from gel filtration chromatography (Fig. 6).

THROMBOPOIETIN RECEPTOR EXPRESSED IN E. coli

FIG. 8. Overlay of sensorgram showing the binding/release of different concentrations (16.9 nM, 33.9 nM, 67.8 nM, 135.6 nM, 169.5 nM) of rhTPOR-EN to/from immobilized rhTPO.

peak (fractions 1 and 2) in Fig. 4 eluted with 5 mM imidazole contained an unknown protein; the second peak (fractions 3 and 4) eluted with 24.9 mM imidazole contained a trace amount of rh TPOR-EN with an apparent molecular mass of 59 kDa; and the third peak (fractions 6 –10) eluted with 154.25 mM imidazole contained a relatively large amount of rh TPOR-EN. Using this single-step metal affinity chromatography, approximately 3 mg of rh TPOR-EN was reproducibly obtained from 1 L of bacterial culture (Table 1) with approximately 80 – 85% homogeneity based on Coomassie blue staining. However, a smaller sized contaminant appeared at all purification stages, which might be a proteolytic breakdown product of rh TPOR-EN based on its smaller molecular size and similar binding affinity. The result of refolding of the denatured rh TPOR-EN by gel filtration chromatography and stepwise gradient is shown in Fig. 6. The rh TPOR-EN was eluted as a single sharp peak. The molecular mass of the eluted protein was in agreement with the calculated value from the amino acid sequence of rh TPOR-EN (Fig. 7). During the gel filtration chromatography, no protein precipitation was detected. This could be due to a separation between the folded protein and the undesired partially folded intermediates. At the time of this study, no commercial anti-human TPOR antibody was available to assess the tertiary state of our rh TPOR-EN product. Therefore, far UV CD was performed to check if our rh TPOR-EN product was in a refolded state. The result indicated that the product had a single minimum and maximum peak at 205 and 192.5 nm, respectively. Based on the CD spectrum, it was estimated that our product contained 0.6% ␣-helix, 67.5% ␤-sheet, 1.3% turn, and 30.7% random structure. Thus the purified rh TPOR-EN was in a

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refolded state, with a predominantly ␤-sheet structure which is characteristically consistent with the hematopoietic cytokine receptor family (3). The biochemical function of the refold rh TPOR-EN was demonstrated by its specific binding to rh TPO as a ligand in a biosensor. The real time sensorgram with a change in RU over time (Fig. 8) showed a strongly concentration-dependent binding of the rh TPOR-EN to the surface-immobilized rh TPO in the concentration range of 16.9 to 169.5 nM. No nonspecific binding was evident when BSA was used as a negative control (data not shown). The association (k ass) and dissociation (k diss) rate constants were calculated to be 0.0072 M ⫺1 s ⫺1 and 10,900 s ⫺1, respectively, and the overall affinity (k D) was 6.6 ⫻ 10 ⫺7 M. We therefore concluded that it is feasible to use an E. coli expression system for production of rh TPOR-EN. We have thus demonstrated, for the first time, a production and purification scheme that is useful for the production of sufficient quantities of rh TPOR-EN for further structural and functional studies. ACKNOWLEDGMENTS The authors thank Dr. Wei Wang in the Biochemistry Department of Hong Kong University of Science and Technology and Mr. Paul Tsang at the Chinese University of Hong Kong for their help in performing the far-UV circular dichroism spectroscopy and the surface plasmon resonance experiments, respectively. Christine K. F. Ng is supported by a Postgraduate studentship provided by The Hong Kong University Granting Council.

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