Expression, purification, ancod characterization of a novel rembinant fusion proteinTPOSCF, Ecoli.

Protein Expression and Puri W cation 47 (2006) 427–433

https://www.360docs.net/doc/e23537925.html,/locate/yprep

1046-5928/$ - see front matter ? 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.pep.2005.10.024

Expression, puri W cation, and characterization of a novel recombinant

fusion protein, rhTPO/SCF, in Escherichia coli ?

Yuhui Zang a , Xu Zhang a,b , Dawen Yuan a , Yumin Zhang a , Jie Zhu a ,

Haiqin Lu a , Chao Chang a , Junchuan Qin a,¤

a

State Key Laboratory of Pharmaceutical Biotechnology, School of Life Science, Nanjing University, Nanjing 210093,PR China b

Institute of Agrobiological Genetics and Physiology, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, PR China

Received 29 September 2005, and in revised form 21 October 2005

Available online 16 November 2005

Abstract

Thrombopoietin (TPO) is the principal regulatory cytokine of megakaryopoiesis and thrombopoiesis and promotes all aspects of megakaryocyte development. Stem cell factor (SCF) is mainly a pleiotropic cytokine acting on hematopoiesis by promoting the survival and proliferation of hematopoietic stem cells and has a potent synergistic e V ect on megakaryopoiesis in the presence of TPO. Here, we report the construction, expression, and puri W cation of a novel recombinant human thrombopoietin/stem cell factor (rhTPO/SCF) fusion protein, which consists of a truncated human thrombopoietin (1–157 a.a.) plus a truncated human stem cell factor (1–145 a.a.), linked by a peptide (G G G G SPG G SG G G G SG G ). The TPO/SCF gene was cloned into the Escherichia coli expression vector pET28a and expressed in BL21(DE3) strain. The rhTPO/SCF constituted up to 6% of the total bacterial protein. Co-expression with E. coli chaper-ones, Trigger Factor (TF) and GroES/GroEL, and lowering cultivation temperature cooperatively improved the solubility of expressed rhTPO/SCF, resulting in about fourfold increase in the yield soluble rhTPO/SCF. The rhTPO/SCF was puri W ed to homogeneity using anion exchange followed by metal a Y nity chromatography. Western blot analysis con W rmed the identity of the puri W ed protein. rhTPO/SCF stimulated a dose-dependent cell proliferation in both TF1 and Mo7e cell lines.? 2005 Elsevier Inc. All rights reserved.

Keywords:Megakaryopoiesis; Thrombopoietin; Stem cell factor; Fusion protein; Chaperone

Megakaryocyte development is a complex process that occurs through multiple interactions with cytokines, extra-cellular matrix proteins, and neighboring cells in the bone marrow environment [1,2]. The e V ects of various combina-tions of cytokines on di V erent aspects of megakaryopoiesis have been studied extensively over the last few years.Among these cytokines, thrombopoietin (TPO)1 was char-acterized to be the principal regulatory cytokine [3–5] and promote all aspects of megakaryocyte development [6,7].

Apart from its e V ects on progenitor cells of megakaryocyte lineage, TPO has also been shown to promote the survival of hematopoietic stem cells [8,9]. Mature human TPO is a single 332 amino acid protein consisting of two domains.The amino terminal W rst 153 amino acids of TPO contain the entire receptor-binding region and the carboxyl termi-nal domain of the molecule contains N-linked glycosylation sites and functions to promote the process of TPO secretion and increase the circulatory survival of the protein [10].Hematopoietic stem cells are characterized by their abil-ity to both self-renew and di V erentiate, and thus give rise to various kinds of mature hematopoietic cells including megakaryocyte [11]. Stem cell factor (SCF) acts on hemato-poiesis by promoting the survival, proliferation, and di V er-entiation of hematopoietic stem cells and progenitor cells by itself or synergizing with other cytokines, resulting in a

?

The project was supported by the National Natural Science founda-tion of China (Grant No. 30270703) to Junchuan Qin.*

Corresponding author. Fax: +86 25 83324605.E-mail address: jcqin@https://www.360docs.net/doc/e23537925.html, (J. Qin).1

Abbreviations used: TPO, thrombopoietin; SCF, stem cell factor;rhTPO, recombinant human thrombopoietin; TF, trigger factor.

428Y. Zang et al. / Protein Expression and Puri W cation 47 (2006) 427–433

synergistic enhancement of adequate expansion and devel-opment of the hematopoietic lineages [12,13]. Domen and Weissman [14] found that SCF and BCL-2 were two sepa-rate signals to prevent apoptosis in hematopoietic stem cells. Although SCF alone does not support megakaryocyte colony formation, it has been shown to have a potent syner-gistic e V ect on megakaryopoiesis in the presence of TPO [15,16]. Furthermore, in the presence of stem cell factor, TPO induced self-renewal of hematopoietic stem cells more e Y ciently than IL-3 or IL-6 did [17]. SCF exists naturally as membrane-anchored and soluble isoforms as a result of alternative RNA splicing and proteolytic processing [18]. The soluble form of SCF has 165 amino acids, and the W rst 141 residues are necessary for SCF activity [19].

Constructions and administration of fusion hematopoi-etic cytokines with multi-activities have been proved to be more e V ective than a single cytokine in the hematopoietic cell proliferation and o V ered some successful applications such as PIXY321, a GM-CSF and IL-3 fusion protein, in medical therapy [20,21]. Providing the pronounced e V ects of SCF on hematopoietic stem cells, the combined adminis-tration of TPO and SCF would be expected to exhibit more potential ability in preserving or preventing the exhaustion of progenitors during the process of megakaryopoiesis and thrombopoiesis. In the present study, a novel TPO/SCF fusion protein was constructed to combine the complemen-tary biologic e V ects of these two cytokines (TPO and SCF) into a single molecular with multilineage activity. The fusion protein consists of a truncated TPO (1–157 a.a.) and a truncated soluble SCF (1–145 a.a.), linked head to tail by a short peptide (GGGGSPGGSGGGGSGG). The rhTPO/ SCF expressed in and puri W ed from Escherichia coli cells exhibited distinct speci W c activities to stimulate a dose-dependent proliferation in both TF1 and Mo7e cell lines. Materials and methods

Materials

Restriction enzymes, Klenow fragment of DNA polymer-ase I, and T4 DNA ligase were from New England BioLab (Beverly, CA, USA). Taq DNA polymerase was from TaKaRa (Dalian, China). DEAE–Sepharose Fast Flow was from Amersham Pharmacia Biotech (Uppsala, Sweden). IPTG was from Sino-American Biotechnology (Shanghai, China). Micro BCA protein assay reagent was from Pierce (Rockfold, IL, USA). The 6-histidine (Epitope Tagging) AB-1 (Clone 4D11) mouse monoclonal antibody was from Lab Vision (Fremont, CA, USA). Antibiotics and protein molecular weight markers were from BBI Fermentas (Toronto, Canada). rhSCF was from New England BioLab (Lake Charles, LA, USA) and rhTPO was from PeproTech (Rocky Hill, NJ, USA). Plasmids and bacteria strain

The plasmid pUC18-SCF containing full length human soluble SCF cDNA was kindly provided by Professor Beifen Shen from Academy of Military Medical Science, China [22]. The plasmid pUC18-TPO containing human TPO cDNA was kindly provided by Dr. Xueyuan Jiang from Nanjing University, China. The plasmid pG-Tf2, which could express E. coli molecular chaperones trigger factor and GroEL/GroES together under the tetracycline-inducible promoter Pzt-1 was generously provided by Dr. Hideki Yanagi from HSP Research Institute, Japan. The plasmid pET32a, pET28a, and host strain BL21 (DE3) was from Novagen.

Cells

Erythroleukemic cell line TF1 [23] and megakaryoblastic cell line Mo7e [24] were both generously provided by Pro-fessor Beifen Shen from Academy of Military Medical Sci-ence, China. TF1 was maintained in DMEM supplemented with 10% FBS and 3ng/ml IL-3. Mo7e was maintained in DMEM supplemented with 10% FBS and 20ng/ml G M-CSF.

Cloning of TPO/SCF gene and construction of its expression plasmid

The TPO/SCF gene cloning and its expression plasmid construction strategies were shown in Fig.1. Brie X y, the DNA fragment encoding part of the linker peptide (G SG G G G SG G) and truncated human SCF (1–145 a.a.) was ampli W ed from the plasmid pUC18-SCF by PCR with a sense primer (5? CC GGATCC GGAGGAGGAGGCTCC G G CG G CG AAG G G ATCTG CAG G AATCG T 3?) and an antisense primer (5? G G AAGCTT ACTTAATGTTGA AGAAAC 3?). The PCR was carried out with 2.5U of pfu DNA polymerase (TaKaRa) in a W nal volume of 50 l using the following conditions: 94°C for 30s, 54°C for 30s, and 72°C for 90 s for 30 cycles. The PCR product was dou-ble digested with Bam HI and Hin dIII, puri W ed by agarose gel electrophoresis, and cloned into pBluescript to yield pBluescript-3?SCF. The DNA fragment encoding truncated human TPO (1–157 a.a.) and the other part of the linker peptide (G G G G SPG) was ampli W ed from the plasmid pUC18-TPO by PCR with a sense primer (5? CC TCTAG A TCTAGCCCGG CTCCTCCTG CT 3?) and an antisense primer (5? CG CCCG G G G AACCTCCTCCTCCG G G TG G G G CCCG CCTG AC 3?) as above. The PCR products were digested with Xba I, puri W ed by agarose gel electropho-resis, and cloned into pBluescript-3?SCF between Xba I and Bam HI (Klenow W lled the Bam HI site to blunt before Bam HI digestion) to yield pBluescript-TS. The plasmid was then sequenced to con W rm the TPO/SCF coding region by using an automatic sequencer (Applied Biosystems model 377). The pBluescript-TS was digested with Bgl II and Hin-dIII and the released TPO/SCF DNA fragment was inserted into the expression vector pET32a at Bam HI and Hin dIII site to yield pET32a-TSH. Finally, the pET32a-TSH was double digested with Nco I and Hin dIII and the released fragment was cloned into expression plasmid

Y. Zang et al. / Protein Expression and Puri W cation 47 (2006) 427–433429

pET28a at Nco I and Hin dIII sites to yield pET28a-TSH. In pET28a-TSH, the TPO/SCF coding region was located immediately upstream of 6-histidine tag coding sequence and stop codon in the same reading frame.Expression of rhTPO/SCF

The expression plasmid pET28a-TSH was transformed into E. coli BL21 (DE3). Single colony was inoculated into fresh liquid LB medium containing 34mg/L kanamycin.Expression of rhTPO/SCF was induced by addition of IPTG to a W nal concentration of 0.2mM, when the culture was grown to OD 600D 0.8. Three hours after induction, the culture was collected and analyzed by 12% SDS–PAG E.The Coomassie-stained SDS–PAGE gel was scanned with a UVP white/ultraviolet transilluminator and protein band was quanti W ed with Grab-it 2.5 and Gelwork software.

Co-expression of GST–VAS with chaperones

Plasmid pG-Tf2, which could express E. coli molecular chaperones trigger factor (TF), and G roEL/G roES together under the tetracycline-inducible promoter Pzt-1,contains a chloramphenicol resistance gene and is compati-ble with plasmid with ColE1 origin [25,26]. The plasmid

pET28a-TSH was transformed into E. coli BL21 (DE3)together with pG-Tf2 and positive clones were selected on solid LB medium containing kanamycin (34mg/L) and chloramphenicol (20mg/L). The transformant was grown at 37°C in fresh LB medium containing 34mg/L kanamy-cin and 20mg/L chloramphenicol. When OD 600 was around 0.8, the culture was transfer to 27°C and continued for half an hour incubation. Then, tetracycline was added to a W nal concentration of 20 g/L to induce chaperones expression and IPTG was added 20min later to induce the expression of the recombinant protein at the W nal concentration of 0.2mM. After induction, the culture was incubated for an additional 5h at 27°C before collection.Puri W cation of rhTPO/SCF

After one liter bacterial culture was grown and induced as described above, the cells were collected by centrifuga-tion, re-suspended in 50ml pre-cooled bu V er A (20mM Tris–HCl, pH 7.8) and lysed by ultrasonication. The lysate was centrifuged at 12,000rpm for 30min and the superna-tant was loaded onto DEAE–Sepharose Fast Flow column (Amersham Pharmacia) equilibrated with bu V er A. After washing the column to baseline with bu V er A, the target protein was eluted step-wise with bu V er A containing di V er-ent concentration of NaCl (50, 100, 200, 300, and 500mM)at 1ml/min. The eluted fraction containing 6￡His-tagged rhTPO/SCF was dialyzed against water and freeze-dried.Then, the sample was dissolved in 10ml ice-cold bu V er B (20mM Tris–HCl, pH 7.9, 100mM NaCl, and 5mM imid-azole) and loaded onto 2ml bed volume of Ni-charged His ·Bind resin column (Novagen). After washing the column sequentially with bu V er B and bu V er C (20mM Tris–HCl, pH 7.9, 100mM NaCl, and 20mM imidazole),the target protein was then eluted with bu V er D (20mM Tris–HCl, pH 7.9, 100mM NaCl, and 200 imidazole) and dialyzed immediately against bu V er E (20mM Tris–HCl,pH 7.9). The column was stripped with strip bu V er (20mM Tris–HCl, pH 7.9, 100mM NaCl, and 100mM EDTA) and regenerated according to the manufacturer’s instructions.Protein quantity assay

The protein concentration was determined by the BCA assay (Pierce, Rockford, IL, USA) according to the manu-facturer’s protocol using bovine serum albumin as a stan-dard. Fractions collected from the chromatography steps were analyzed on a 12% SDS–polyacrylamide gel.SDS–PAGE and Western blot analyses

The expressed protein was analyzed by reducing SDS–PAG E. All samples were prepared by boiling for 3min in sample loading bu V er containing 10% of 2-mercaptoethanol.The proteins were detected in gels by Coomassie brilliant blue staining or transferred onto a nitrocellulose membrane for Western blot analysis. The Western blot analysis was

Fig.

1. Construction of TPO/SCF DNA fragment and its expression plas-mid (X, Xba I; Ba, Bam HI; Bg, Bgl II; H, Hin dIII; N, Nco I; and His, Histi-dine).

430Y. Zang et al. / Protein Expression and Puri W cation 47 (2006) 427–433

carried out according to the protocol recommended by Bio-Rad (Hercules, CA, USA). Speci W c 6-histidine mouse mono-clonal antibody against 6-histidine was used as the primary

antibody and horseradish peroxidase-conjugated anti-mouse IgG antibody as the detecting reagent.

Cell proliferation assays

Serial twofold dilutions of samples were prepared in 96-well microtiter plate at a volume of 50 l per well. The TF1

or Mo7e cells were then washed three times with PBS and suspended in RPMI 1640 medium containing 20% FCS at W nal concentration of 4.0￡105cells/ml. Fifty microliter ali-quots of the cell suspension were then added to the previ-

ously prepared microtiter plate containing diluted samples. After incubating the microtiter plates at 37°C in fully humidi W ed atmosphere containing 5% CO2 for 48–60h, 10 l of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazo-lium bromide (MTT) stock solution (5mg/ml) was added into each well and the incubation was continued for another 4–6h at room temperature. The insoluble purple reaction product produced by MTT was dissolved by add-ing 100 l of 0.01M HCl–10% SDS solution into each well. After incubating for 16h, the A595 absorbance values were determined on double-channel Microplate Reader. Plot the absorbance at 595nm (Y axis) versus concentration of sam-ple (X axis). The ED50 value was determined as the X axis value that corresponds to one-half the maximum (plateau) absorbance values. The amount of recombinant protein required for half-maximal stimulation of TF-1 or Mo7e cells proliferation was de W ned as one unit.

Results

Construction of rhTPO/SCF gene and its coloning into expression vector

The novel TPO/SCF gene was constructed and coloned into pET28a as described under Materials and methods.Two DNA fragments, encoding a truncated human TPO (1–157 a.a.) and a truncated hSCF (1–145 a.a.), respectively, were designed to be linked in head to tail style by a linker sequence encoding peptide (G G G G SPG G SG G G G SG G). In the recombinant expression plasmid, TPO/SCF gene was incorporated immediately upstream of the 6￡His and stop codon sequence in the same ORF. Thus, the expressed rhTPO/SCF would contain a 6￡His tag at its C terminal. Results of restriction enzyme analysis and DNA sequenc-ing con W rmed the authenticity of TPO/SCF coding region. Expression of rhTPO/SCF

Escherichia coli strain BL21(DE3) cells harboring pET28a-TSH were cultured as described under Materials and methods. After inducing expression by 0.2M IPTG for 3h at 37°C, SDS–PAGE analysis of the cell lysate detected a protein band with molecular weight about 34.5kDa, which corresponded to the calculated molecular weight of recombinant rhTPO/SCF. Under this condition, the expressed protein was about 6% of total bacterial protein as determined by densitometric scanning (Fig.2A). When total cellular proteins were separated into soluble and insol-uble fractions, more than 80% of rhTPO/SCF proteins were found to be present in the insoluble fraction.

For the sake of improving solubility, lowering cultiva-tion temperature and co-expression with molecular chaper-ones, TF and GroEL/GroES, were performed as described under Materials and methods. Neither lowering cultivation temperature nor molecular chaperones had signi W cant e V ects on the yield of soluble rhTPO/SCF independently (data not shown). However, combination of these two kinds of e V orts exhibited pronounced e V ects on improving rhTPO/SCF solubility. Growing culture at 27°C and induc-ing expression of molecular chaperones with tetracycline at

a W nal concentration of 20 g/L, followed by induction with

0.2mM IPTG, were optimized as suitable expression condi-tions for high level production of soluble and biologically active recombinant TPO/SCF. When rhTPO/SCF was

Fig.2. (A) SDS–PAGE analysis of rhTPO/SCF. Lane M, protein molecular mass markers (kDa); lane 1, the cell lysate of non-induced BL21(DE3) con-

taining the recombinant expression plasmid pET28a-TSH; lane 2, the cell lysate of IPTG induced BL21(DE3) containing the recombinant expression plas-mid pET28a-TSH. (B) Yield of soluble rhTPO/SCF under di V erent expression conditions. To determine the quantity of soluble rhTPO/SCF, TF1 cells were incubated with various volume of supernatant of cell lysate. In two parallel experiments, 100ml culture with same cell density was used in the expres-sions of rhTPO/SCF with co-expression of molecular chaperones (?) at 27°C or without co-expression of molecular chaperones (?) at 37°C as described under Materials and methods. After cells were collected, the cell pellet was re-suspended in 10ml of 1￡PBS bu V er and lysed by ultrasonication.

Y. Zang et al. / Protein Expression and Puri W cation 47 (2006) 427–433431

co-expressed with TF and G roEL/G roES at 27°C, the amount of rhTPO/SCF appeared in the soluble fraction increased about fourfold than that expressed at 37°C with-out chaperons, according to their respective potentials to stimulate TF1 cell proliferation (Fig.2B).Puri W cation of rhTPO/SCF

The puri W cation procedure of rhSCF/M-CSF is described under Materials and methods and the result is summarized in Table 1. The rhTPO/SCF protein was puri-W ed to near homogeneity from soluble fraction of the cells by combination of anion exchange chromatography and an a Y nity chromatographic step using Ni-charged His ·Bind resin. An extensive puri W cation was achieved by DEAE–Sepharose Fast Flow column, as indicated both from the SDS–PAGE result (Fig.3A) and bioactivity assays (Table 1).Then, rhTPO/SCF was further puri W ed by Ni-charged a Y n-ity chromatography, where its elution showed a single band on SDS–PAG E with an apparent molecular weight of 34.5kDa as expected. From 1L culture, a total of 4.8mg puri W ed protein was obtained. Western blot analysis con-W rmed the identity of the protein and no degradation frag-ment was detected (Fig.3B).Cell proliferation assay

rhTPO/SCF promoted a dose-dependent proliferation response on both erythroleukemic cell line TF1 and mega-karyoblastic cell line Mo7e. Previous report had shown that Mo7e cells had a high rate of proliferation when cultured in the presence of TPO but proliferated poorly in the presence of SCF alone [27]. With respect to TF1, it exhibited a dose-dependent proliferation to the stimulations of G M-CSF,Il-3, and SCF but was not sensitive to TPO [23,28]. Thus,they could be used to determine the activity of SCF and TPO, respectively. The ED 50 of puri W ed rhTPO/SCF was about 5.0ng/ml in TF1 cell proliferation assay, correspond-ing a speci W c activity of 2.0￡105U/mg, while the ED 50 of rhSCF from E. coli was 2.8ng/ml under the same experimen-tal conditions. The ED 50 of puri W ed rhTPO/SCF was about 1.2ng/ml in Mo7e cell proliferation assay, corresponding a speci W c activity of 8.3￡105U/mg, while the ED 50 of rhTPO (1–158 a.a.) from E. coli was about 1.0ng/ml.

Discussion

Thrombocytopenia is a common problem in oncology and hematology. In past two decades, the availability of myeloid and erythroid cytokines has provided valuable therapeutic agents in management of the disease. Several cytokines, including TPO, IL-3, IL-6, IL-11, and PIXY321,have ever been evaluated in preliminary clinical trials [29,30]. Among them, TPO is clearly the most potent one to stimulate megakaryopoiesis and promote platelet recovery.Nevertheless, despite its initial rapid demonstration of a strong thrombopoietic activity, rhTPO still remains under investigation to date. TPO and SCF are functionally related cytokines with overlapping but distinct hematopoietic e V ects. Their complementary biologic e V ects on megak-aryopoiesis prompted us the attempt to construct a TPO/SCF fusion protein. The design of TPO/SCF fusion protein was aimed to combine and enhance the synergistic e V ects between SCF and TPO. Thus, the novel fusion protein could exert its e V ects more e Y ciently on preserving or pre-

Table 1

Puri W cation of rhTPO/SCF from E. coli BL21(DE3)a a One liter of culture was used in this preparation and the total cell extract was collected from approximate 7.5g wet weight cells.b Total protein was determined by BCA assay.

c Total activity of the sample was determine

d by th

e ability to stimulate TF1 cell proliferation.

d Estimated protein product was calculated by dividing th

e total activity in each step by the speci W c activity o

f puri W ed rhTPO/SCF.e

Approximate purity was determined by densitometric scanning.

Puri W cation stage

Total protein (mg)b Total activity (U)c Estimated rhTPO/SCF product (mg)d Approximate purity of rhTPO/SCF (%)e Recovery (%)Total cell extract e

225 2.10￡10610.5 4.6100DEAE–Sepharose Fast Flow 41 1.54￡1067.719.173Ni-charged His ·Bind resin

5.2

0.96￡106

4.8

92.5

62

Fig.

3. SDS–PAGE analysis of rhTPO/SCF at di V erent stages of puri W ca-tion and Western blot analysis following puri W cation. (A) Samples of rhTPO/SCF were electrophoresed on 12% denaturing polyacrylamide gel and stained with Coomassie blue; (B) Western blot analysis of puri W ed rhTPO/SCF. The protein was detected with mouse monoclonal antibody against 6-histidine as described under Materials and methods. Lane 1, the elution from DEAE–Sepharose Fast Flow column; lane 2, rhTPO/SCF eluted from Ni-charged His ·Bind resin column; lane M, protein molecu-lar mass markers (kDa).

432Y. Zang et al. / Protein Expression and Puri W cation 47 (2006) 427–433

venting the exhaustion of progenitors during the process of megakaryopoiesis and thrombopoiesis.

In the present study, rhTPO/SCF was designed to con-sist of two moieties linked in head to tail style by a peptide (GGGGSPGGSGGGGSGG). The linker peptide was rel-atively non-antigenic determinant and X exible to enable that the two subunits could wiggle freely and maintain their three-dimensional structures. In our previous studies [28,31], we had successfully constructed SCF/M-CSF and dual SCF fusion proteins, respectively, with a structurally similar linker peptide (G G G G SG G G G SG G), showing that both moieties of the fusion proteins could bind to their receptors independently. In case of TPO/SCF, we added a proline residue in the linker peptide. Computer assistant design revealed that addition of proline residue would increase the distance between two moieties within the fusion molecule, facilitating TPO and SCF moieties to bind their receptors independently (data not shown). This may have important clinical implications for alleviat-ing thrombocytopenia, because stem cell enrichment with megakaryocytic progenitors was considered to improve the recovery of platelets after myeloablative therapy [32–34].

An initial attempt was made to express rhTPO/SCF from the recombinant plasmid pET32a-TPO/SCF (Fig.1), in which the TPO/SCF DNA fragment was cloned into pET32a and fused with thioredoxin tag. SDS–PAGE analy-sis showed that the amounts of the target protein could reach about 25% of the total cell lysate. However, the majority of target protein was insoluble and further West-ern blot analysis revealed that multiple degradation frag-ments were present in the soluble fraction (data not shown). We had tried di V erent methods to refold thioredoxin-TPO/ SCF inclusion bodies in vivo, but the yield of soluble and biologically active rhTPO/SCF was very low.

Here, we reported construction and characterization of the novel recombinant human TPO/SCF fusion protein. The availability of large amounts of pure and active rhTPO/SCF could facilitate further studies on its in vivo activities in pro-moting megakaryopoiesis and platelet recovery. Acknowledgments

The authors are grateful to Dr. Hideki Yanagi and Dr. Hua Zichun for kindly providing plasmid pG-Tf2. References

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