Protein Expression and Purification 105 (2015) 1–7

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Expression of bioactive soluble human stem cell factor (SCF) from recombinant Escherichia coli by coproduction of thioredoxin and efficient purification using arginine in affinity chromatography Teruo Akuta a,b,⇑, Takane Kikuchi-Ueda a,⇑, Keitaro Imaizumi a,b, Hiroyuki Oshikane c, Toshio Nakaki c, Yoko Okada d, Sara Sultana d, Kenichiro Kobayashi d, Nobutaka Kiyokawa d, Yasuo Ono a a

Department of Microbiology and Immunology, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan Kyokuto Pharmaceutical Industrial Co. Ltd., 7-8, Nihonbashi Kobunacho, Chuo-ku, Tokyo 103-0024, Japan c Department of Pharmacology, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan d Department of Pediatric Hematology and Oncology Research, National Research Institute for Child Health and Development, 2-10-1, Okura, Setagaya-ku, Tokyo 157-8535, Japan b

a r t i c l e

i n f o

Article history: Received 28 August 2014 and in revised form 19 September 2014 Available online 5 October 2014 Keywords: Human stem cell factor Escherichia coli Soluble protein expression Affinity tag-based protein purification L-Arginine

a b s t r a c t Stem cell factor (SCF) known as the c-kit ligand is a two disulfide bridge-containing cytokine in the regulation of the development and function of hematopoietic cell lineages and other cells such as mast cells, germ cells, and melanocytes. The secreted soluble form of SCF exists as noncovalently associated homodimer and exerts its activity by signaling through the c-Kit receptor. In this report, we present the high level expression of a soluble recombinant human SCF (rhSCF) in Escherichia coli. A codon-optimized Profinity eXact™-tagged hSCF cDNA was cloned into pET3b vector, and transformed into E. coli BL21(DE3) harboring a bacterial thioredoxin coexpression vector. The recombinant protein was purified via an affinity chromatography processed by cleavage with sodium fluoride, resulting in the complete proteolytic removal the N-terminal tag. Although almost none of the soluble fusion protein bound to the resin in standard protocol using 0.1 M sodium phosphate buffer (pH 7.2), the use of binding buffer containing 0.5 M L-arginine for protein stabilization dramatically enhanced binding to resin and recovery of the protein beyond expectation. Also pretreatment by Triton X-114 for removing endotoxin was effective for affinity chromatography. In chromatography performance, L-arginine was more effective than Triton X114 treatment. Following Mono Q anion exchange chromatography, the target protein was isolated in high purity. The rhSCF protein specifically enhanced the viability of human myeloid leukemia cell line TF-1 and the proliferation and maturation of human mast cell line LAD2 cell. This novel protocol for the production of rhSCF is a simple, suitable, and efficient method. Ó 2014 Elsevier Inc. All rights reserved.

Introduction Stem cell factor (SCF) is a dimeric molecule that exerts numerous effects on a broad range of cell types by activating the receptor tyrosine kinase c-Kit [1]. SCF is produced by endothelial cells, fibroblasts, keratinocytes, gut epithelial cells and tumor cells [2,3]. SCF can act on hematopoiesis by stimulating the survival and proliferation of stem cells and progenitor cells [4]. It is also crucial for mast cell proliferation, function [5–7] and the development of other ⇑ Corresponding authors at: Department of Microbiology and Immunology, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan (T. Akuta). E-mail addresses: [email protected] (T. Akuta), takane@med. teikyo-u.ac.jp (T. Kikuchi-Ueda). http://dx.doi.org/10.1016/j.pep.2014.09.015 1046-5928/Ó 2014 Elsevier Inc. All rights reserved.

cells, including melanocytes and germ cells [8]. Some tumor cell proliferation and invasiveness are promoted by SCF [9]. SCF exists naturally as a membrane-bound protein composed of either 220 or 248 amino acids and soluble isoform (SCF164) as a result of alternative RNA splicing and proteolytic processing [9,10,11]. The soluble protein (SCF164), which has two intramolecular disulfide bonds (Cys4-Cys89 and Cys43-Cys138), is a noncovalently associated dimer under non-denaturing conditions [10]. The glycosylated or unglycosylated form does not influence biological activity [7,12]. SCF in clinical trials have been performed in the treatment of anemia [13] and gene therapy to stimulate and expand hematopoietic stem cells (HSCs) [14]. Recombinant human SCF (rhSCF) can maintain hematopoietic stem cells and mast cell in culture [15,16], and it is widely tested in research into differentiation from embryonic stem cell (ESC)/induced pluripotent stem cell (iPSC) [17,18].

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Several cytokines and growth factors including rSCF are commercially available but their limited biological activity and cost hampered development of blood cell and ESC/iPSC research and/ or extended application in clinical area. It would therefore be desirable to have sources of highly purified and biologically active cytokines, including SCF, in large quantities. Methods for the production and purification of rhSCF from Escherichia coli have previously been attempted and reported by many groups. Some groups reported the expression of mature SCF as insoluble inclusion bodies with subsequent refolding procedures [19–26]. Recently Bals et al. [27] reported murine SCF in a thioredoxin based fusion protein as soluble form with subsequent TEV protease cleavage and membrane chromatography. However, most of the commercial and reported rhSCFs derived from E. coli have an extraneous N-terminal L-methionyl residue, thus rSCFs of this type are especially called ‘‘L-methionyl SCF’’. In the present report, we describe a high-level expression and purification process for soluble rhSCF164 in E. coli without any extra amino acid overhang (non-methionyl hSCF). In our approach, rhSCF from a codon-optimized, chemically synthesized gene template is expressed as soluble protein in fusion with N-terminal codon-optimized Profinity eXact™ tag [28] and coexpression of bacterial thioredoxin in E. coli BL21(DE3) [29,30]. One of the advantageous points in using Profinity eXact™ tag strategy is the precise excision at the very C-terminus of the recognition site, which can yield the cleaved product without any additional amino acid overhangs. On the other hand, the coexpression of thioredoxin is known to facilitate disulfide bond formation in the cytoplasm, leading to rigid expression of the target protein harboring SAS bonds. Additionally, we have tested L-arginine for Profinity eXact™ affinity chromatography. Some groups have shown that L-arginine is effective for improving column chromatographic performances [31], e.g., elution of antibodies from Protein A [32,33], reduction of protein associated endotoxin during a wash-step of Protein Abound antibodies [34], reduction of non-specific binding of proteins in gel permeation chromatography [35], improvement of elution recovery in hydrophobic chromatography and dye-affinity chromatography [36,37]. L-Arginine is a normal amino acid in human and animal and has a low order of toxicity. Thus low concentration of L-arginine in final formulation is not a problem for research use, or even for biopharmaceutical application. Here we describe a novel method which enabled the binding and elution recovery hSCF from Profinity eXact™ affinity chromatography with the use of L-arginine. Finally, bioactivity of the purified rhSCF was tested using cultures of human erythroleukemia cell line TF-1 cell and human mast cell line LAD2 cells.

selected on a Luria–Bertani (LB) agar plate with antibiotics (100 lg/mL ampicillin and 34 lg/mL chloramphenicol). For recombinant fusion protein expression, the selected clones were grown overnight in 2 mL of LB with the antibiotics containing 2% glucose in order to inhibit transcription from lac promoter and to prevent leaks of expressed protein. The overnight culture broths were then diluted 1:50 in fresh 2 mL of 2  YT broth containing 2% glucose and antibiotics (2  YTG) and grown at 37 °C until the OD600nm reached approximately 0.6–0.8. Recombinant fusion protein expression was then induced by the addition of isopropyl-b-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM, and the cells were incubated for 3 h at 30 °C with 180 rpm shaking. To determine the expression level of target protein, the total amounts of cell protein were analyzed on 10–20% SDS–polyacrylamide gel (Supersep, Wako, Japan) and detected by Coomassie brilliant blue R350 (GE Healthcare, USA). Next, to generate the recombinant fusion protein in a soluble form, the thioredoxin (Trx)1 coproduction system by using pT-Trx vector was examined. The pT-Trx plasmid, which contains the T7 promoter linked to the E. coli Trx and pACYC-based replicon, was a kind gift from Dr. Shunsuke Ishii of the Laboratory of Molecular Genetics, RIKEN Tsukuba Institute, Japan. The E. coli strains BL21(DE3) harboring pT-Trx or pLysS were transformed by pET3beXact-hSCF. Coproduction of Trx increases the solubility of foreign proteins and was transduced with the Trx expression vector, pTTrx. The cell pellets were washed with 0.1 M sodium phosphate buffer (pH 7.2) and frozen at 80 °C until protein purification.

Affinity purification and proteolytic cleavage of eXact-hSCF fusion protein

Expression of recombinant eXact-hSCF fusion protein

The expressed fusion proteins were purified by the Profinity eXact™ affinity column (Superflow™ crosslinked agarose coupled to a unique subtilisin mutant, Bio-Rad Laboratories, USA). Once prodomain/Profinity eXact fusion protein has been captured by the Profinity eXact™ resin—and the cleavage reaction has been triggered with a sodium fluoride-containing buffer—the affinity tag can be precisely cleaved off at the very C-terminus end of the recognition sequence. As a result, the tag-free recombinant protein is released and eluted. Initially, cell lysis and the following affinity purification were performed according to Bio-Rad’s manual. The frozen cell pellet (about 0.06 g E. coli wet weight from 20 mL culture) was resuspended in 600 ll of 0.1 M sodium phosphate buffer (pH 7.2) containing 1 Bugbuster (Novagen), protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan), and sonicated on ice for 1 min (TOMY SEIKO, Tokyo, Japan) until it was completely fragmented, as observed by microscopic inspection. After centrifugation at 20,000g for 60 min at 4 °C, the supernatant was loaded onto a Mini Bio-Spin columns (Bio-Rad), containing 500 ll of pre-equilibrated resin equilibrated with pre-chilled 0.1 M sodium phosphate (pH 7.2) and incubated with rotating for 20 min at 4 °C. The unbound protein was collected by spinning at 1000g for 1 min. Spin column was washed with 5 ml of 0.1 M sodium phosphate (pH 7.2) over 10 Column Volume (CV). Column was plugged with end cap and incubated in room temperature (RT) in 300 ll of elution buffer (0.1 M sodium phosphate (pH 7.2) containing 0.1 M NaF). After incubation for 30 min, the eluate containing tag-free hSCF was collected by spinning at 1000g for 1 min. The pooled protein (300 ll) was analyzed on SDS–PAGE. Alternatively, two different methods were tested, i.e., non-ionic detergent Triton X-114 used for removing endotoxin before affinity

The construct of pET3b-eXact-hSCF vector was transformed into E. coli BL21(DE3) pLysS (Novagen) and positive clones were

1 Abbreviations used: hSCF, human stem cell factor; Trx, thioredoxin; IPTG, isopropyl-b-D-thiogalactopyranoside; LB, Luria–Bertani.

Materials and methods Construction of the pET3b-eXact-hSCF The synthetic gene coding for N-terminal Profinity eXact™ tagged soluble hSCF encoding 164 amino acids (hSCF164) was designed on the basis of codon preference in E. coli (Biomatik, Canada). A codon-optimized Profinity eXact tag is added 50 to the gene coding for soluble hSCF164. This synthetic nucleotide was digested with NdeI and BamHI restriction enzymes (Takara, Japan) and then subcloned into NdeI/BamHI digested pET3b expression vector (Novagen, USA) to produce the pET3b-eXact-hSCF that encodes the eXact tag-hSCF fusion protein (Fig. 1).

T. Akuta et al. / Protein Expression and Purification 105 (2015) 1–7

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Fig. 1. The synthetic gene was designed as described in Materials and Methods. The codons were selected based on the codon usage preference in E. coli. The amino acid sequence as coded by the synthetic gene is also presented. Profinity eXact tag is added 50 to the gene coding for soluble hSCF. Mature rhSCF (164 aa) was cleaved by engineered protease (subtilisin), allowing the exact excision at the N-terminus of the target rhSCF to yield rhSCF without additional methionine. The arrow indicates the amino terminus of the polypeptide after a protease cleavage. Asterisks represent stop codons. The tandem stop codons are used to prevent read-through.

chromatography [39] and 0.5 M L-arginine added to the buffer for preventing aggregation and reducing endotoxin [34,38]. In a TritonX-114 method, a soluble fraction in 0.1 M sodium phosphate (pH 7.2) was treated with 1% Triton X-114 (Nacalai Tesque) on ice by stirring for 30 min, followed by incubation for 10 min at 25 °C. Subsequently, endotoxin extracted by Triton X-114 was removed in the lower layer by centrifugation 10,000g for 15 min at 25 °C. This procedure was repeated twice and the upper layer fraction was loaded onto a column. Wash and elution was done as described above. In an arginine method, frozen sample was lysed by buffer A (0.1 M HEPES buffer, 0.5 M L-arginine, 0.3 M sodium acetate, 1 mM EDTA), of which pH was adjusted by acetic acid (pH 7.2), containing 1 Bugbuster, and the protease inhibitor cocktail. After centrifugation, a soluble fraction was loaded onto the column and washed with buffer A and the rhSCF eluted from the resin with elution buffer (buffer A containing 0.1 M NaF) for 30 min at RT. Purification of hSCF after proteolytic cleavage Further purification of the eluted fractions after affinity chromatography with L-arginine was performed using Mono Q column 5/ 50 GL (GE Healthcare) at 4 °C on AKTA purifier systems (GE Healthcare). The sample was diluted 10-fold with Tris buffer (50 mM Tris–HCl (pH 7.5)) to the final concentrations of 0.05 M L-arginine and 0.01 M NaF. The column was washed with 10 mL Tris buffer (10 CV) and eluted with a gradient (10 mL) from 0 to 1.0 M NaCl in Tris buffer. Fractions containing hSCF were pooled and concentrated by AmiconÒ Ultra-Centrifugal filter (3 K cut off, Millipore). Purities of the recombinant fusion protein and tag-free protein compared with total protein were quantified by densitometry of the gel band intensities using ImageQuant TL software (GE Healthcare, Japan). In order to further secure the protein sample to cell culture level, yet residual endotoxin was thoroughly removed by 1% Triton X-114 treatment as described above and rhSCF concentration was determined by bicinchoninic acid (BCA) protein assay (PIERCE, Rockford, USA), using bovine serum albumin as a standard.

To test residual endotoxin levels in the final sample, the rhSCF protein solution was analyzed by LAL Test (Toxicolor LS-50M, Seikagaku-kogyo, Japan). Finally, the purified rhSCF protein was filter sterilized (0.2 lm) and stored at 80 °C until further use. Biological activity of purified hSCF protein The functional activities of rhSCF protein were measured by their ability to enhance the viability of human erythroleukemia cell line TF-1 (ATCC) [39]. Cells were routinely grown in RPMI1640 medium (Sigma–Aldrich) supplemented with 5 ng/ml GM-CSF (R&D systems) and 10% fetal calf serum (FCS) (MP Biomedicals, USA). Prior to the SCF biological-assay, actively cycling TF-1 cells were washed twice with PBS to remove all trace of cytokines. The rhSCFs purified as described in this report and commercially available rhSCF (expressed in E. coli, purchased from Peprotech) were incubated, respectively with 1  104 cells in 100 lL of culture medium in a 96-well microplate at 37 °C, 5% CO2 in air at final concentration range from 0.001 to 100 ng/mL. After 72 h of incubation, the growth value was measured by cell ATP content using a commercially available ATP assay kit of cells (TOYO Ink Co., Tokyo, Japan) according to the manufacturer’s instructions. Luciferinluciferase reagent solution (100 lL) was added to each well of a 96-well microplate, and the plate was further incubated for 10 min at 23 °C. End-point luminescence was measured using the FlexStation 3 microplate reader (Molecular Devices Inc.). The effect of hSCF and commercial hSCF on the cells were plotted. The viabilities of the cells were evaluated by the following formula [(Light emission sample  Light emission control)/Light emission control]  100 (%)]. For a negative control, the cells were cultured in the absence of rhSCF. The effective concentration 50% (EC50) was determined by the dose needed for 50% of the maximum effect (biological activity). The data represent mean ± SD of three independent experiments. Human mast cell line LAD2 [16] was obtained from the National Institute of Health and maintained in serum-free medium StemPro-34 (Life Technologies) supplemented with 100 ng/ml

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commercial rhSCF. The rhSCFs purified as described in this report and commercial rhSCF were each incubated with 5  103 cells in 100 lL of culture medium in a 96-well microplate at 37 °C, 5% CO2. After 7 days of incubation, LAD2 cells were attached to glass slides and stained by using Alcian blue-Safranin staining [40]. In order to check the cell morphology, the slides were magnified by using a high resolving microscope (BX53, Olympus). Results

BL21(DE3)pLysS IPTG M W

(kDa) 220-

+ W

BL21(DE3)pT-Trx

+ + S I

- + W W

+ + S I

12010080-

Construction of hSCF fusion protein expression vectors To obtain high-level expression, the hSCF164 gene with codon sequences optimized for E. coli overexpression was cloned into pET3b, which greatly facilitated the rigid production of Profinity eXact™ fusion-tagged hSCF protein (Fig. 1). Expression of the resulting fusion protein eXact-hSCF (with a predicted molecular weight of 28 kDa) is driven by the strong T7 promoter.

60504030-

Soluble expression of eXact-hSCF fusion protein In order to acquire soluble proteins, we performed an expression experiment using E. coli BL21(DE3) with alternatively harboring pLysS or thioredoxin coexpression vector, pT-Trx. As revealed by SDS–PAGE analysis, eXact-hSCF fusion protein (28 kDa) was mainly expressed in the insoluble fraction in BL21(DE3)pLysS (lane 5) (Fig. 2). The solubility of fusion protein was dramatically increased by coproduction of thioredoxin in BL21(DE3)pT-Trx (lane 8) (Fig. 2). These results clearly showed that the E. coli strain BL21(DE3)pT-Trx is a preferred host for the expression of soluble hSCF. According to densitometric scanning results and evaluation of BCA protein assay, eXact-hSCF accumulated up to about 17% of total bacterial proteins and approximately 30–50 mg of soluble forms was produced per liter of culture (Table 1). Purification of hSCF Initially, in accordance with the Bio-rad Profinity eXact™ affinity chromatographic purification manual, we used 0.1 M sodium phosphate buffer (pH 7.2) for binding. However, Almost none of the fusion protein bound to the resin and was observed in flow through fraction (lane 3–5) (Fig. 3A). Next, we explored new binding conditions and tested two methods, i.e., the treatment of 1% Triton X-114 and 0.5 M L-arginine, in an aim for removing endotoxin derived from E. coli and preventing protein aggregation. Beyond expectation, these treatments successfully allowed efficient binding to the resin (lanes 4 and 10) (Fig. 3B). After subtilisin activating by sodium fluoride, the subtilisin prodomain/ Profinity eXact™ tag (8.2 kDa) can be proteolytically removed to yield only untagged, non-methionyl hSCF molecules (164 amino acids residues, 18.5 kDa) (lane 6 and 12) (Fig. 3B). Recovery rate of untagged rhSCF with 0.5 M L-arginine was found to be higher than that by Triton X-114 treatment. Moreover, washing with L-arginine was shown to better reduce contaminant proteins than Triton X-114. Fractions containing rhSCF were further purified by MonoQ anion exchange chromatography (Fig. 3C). Regardless of either reducing or nonreducing conditions on SDS–PAGE sample preparation, only 18.5 kDa rhSCF band appeared, suggesting that rhSCF contained only intramolecular disulfide bonds which should be formed naturally, but no intermolecularly disulfide linked dimers or oligomers which might be formed due to experimental artifact. Final purity was estimated to be greater than 95% by optic densitometry of SDS–PAGE gels (Table 1). The concentration of endotoxin was an undetectable level (1  105 units/mg. This specific activity was slightly superior to that of commercially available rhSCF, which is 10 ng/mL. The biological properties of the purified rhSCF were further tested via examination of the cell differentiation of human mast cell LAD2 cells after the long-term assay. LAD2 cells cultivated

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T. Akuta et al. / Protein Expression and Purification 105 (2015) 1–7 Table 1 Purification of hSCF fusion proteins overexpressed in BL21(DE3)pT-Trx E. coli. Purification step

Total proteina (mg)

hSCF fusion protein (tag-free protein)a,b (mg)

Purityb (%)

Yieldc (%)

Cell lysate Crude extract Affnity chromatography (+ L-Arg) Mono Q ion exchange chromatography

65 53 5 2

11 (7.5) 10 (6.8) (4) (2)

17 18 80 95

100 91 53 26

a

Results are derived from 1 g wet cell weight from 300 mL of culture. Protein concentrations determined by BCA protein assay using BSA as a standard. Fusion protein (26 kDa) and tag-free protein (18 kDa) as estimated by PAGE densitometry with ImageQuant TL (GE Healthcare). The yield is the amount of target protein at that step divided by the amount of target protein in the first step (defined as 100%) and the yield of tag-free protein was recalculated as tagged fusion protein. b

c

A (kDa)

0.1M NaPi (7.2) W S FL Wash E

M

22515010276-

B

Triton X-114 M W S

(kDa)

C

0.5 M L-Arg

FL Wash E

W S

FL Wash E

52-

5238-

31-

31-

24-

24-

17-

17-

12-

12-

S

FL



260140-

22515010276-

38-

M

(kDa)

957252423426-

*

*

17-

10-

1

2

3

4

5

6

1

2

3

4

5

6

7

8

9

10

11

12

1

2

3

4

5

Fig. 3. The expression and purification of rhSCF. (A) Summary of the hSCF purification process in Profinity eXact™ standard protocol. Lane 1, molecular weight marker; Lane 2, total cell protein from BL21(DE3)pT-Trx /pET3b-eXact-hSCF; Lane 3, soluble protein from the supernatant of lysed and centrifuged cells; Lane 4, nonbinding protein after Profinity eXact™ chromatography; Lane 5, washing fraction; Lane 6, products after proteolytic cleavage: the asterisk indicates rhSCF. (B) Binding and recovery of hSCF from Profinity eXact™ resin by using Triton X-114 or L-arginine. Lane 1, molecular weight marker; Lane 2, total cell protein from E. coli; Lane 3, soluble protein from the upper aqueous phase after Triton X-114 treatment; Lane 4, nonbinding protein; Lane 5, washing fraction; Lane 6, products after proteolytic cleavage; Lane 7, empty well with 1 SDS buffer; Lane 8, total cell protein from E. coli; Lane 9, soluble protein from the supernatant using lysis buffer containing 0.5 M L-arginine; Lane 10, nonbinding protein; Lane 11, washing fraction; Lane 12, products after proteolytic cleavage. (C) Purified rhSCF after proteolytic cleavage. Lane 1, molecular weight marker; Lane 2, soluble protein from the supernatant using lysis buffer containing 0.5 M L-arginine; Lane 3, nonbinding protein on affinity chromatography: Lanes 4 and 5 (reducing and nonreducing, respectively), purified rhSCF by Mono Q anion exchange chromatography after proteolytic cleavage.

with the purified rhSCF at 100 ng/mL exhibited the heparin-rich granular cytoplasm (Fig. 5). In contrast, LAD2 cells cultivated without hSCF showed chondroitin sulfate-rich (or immature) granular cytoplasm.

Discussion In this report, we describe a novel method to produce soluble recombinant human stem cell factor (SCF164) in E. coli for highlevel expression with simple purification procedure. We combined several technologies: codon-optimized synthetic gene for high expression, E. coli host BL21(DE3) and thioredoxin coexpression system to increase the solubility of foreign proteins, and Profinity eXact™ tag for simple purification with L-arginine. Laboratory scale conventional shaking cultures and eXact-tag based purification resulted in approximately 6 mg of purified rhSCF per 1 L culture medium. This final yield of the product without a need for refolding step is the highest reported to date. We applied codon optimization by using synthetic gene technology. Recent advances of gene synthesis technologies enable

the rapid acquisition of desirable gene sequences at lower cost. We have achieved a high ratio of soluble to insoluble fusion protein expression by using E. coli strain, BL21(DE3) pT-Trx that coexpresses thioredoxin. Thioredoxin is a soluble cytoplasmic protein with a molecular weight of only 12 kDa. Our results showed that thioredoxin in BL21(DE3) could greatly improve the solubility of fusion protein when compared to BL21(DE3) pLysS. Thioredoxin has been shown to enhance the solubility of recombinant protein and maintain disulfide bond formation in adequate host strains [29,30]. Up to the present, human SCF expression in E. coli has always led to insoluble inclusion bodies, thus the formation of soluble human SCF in this study is obviously due to the coexpression of thioredoxin. The Profinity eXact™ tag system can perform both purifying mature protein and removing their tag in elution step at the same time. This system is based on the immobilized mutant subtilisin protease which recognizes and binds the affinity tag (prodomain of the subtilisin) fused to the N-terminus of the target protein. The cleavage reaction has been triggered with a sodium fluoridecontaining buffer and the affinity tag is precisely cleaved at the C-terminus of a nine amino acid sequence (EEDKLFKAL). By this

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T. Akuta et al. / Protein Expression and Purification 105 (2015) 1–7

Cell viability (%)

100

75

50

25

0 0.001

0.01

0.1

1

10

100

Protein concentration (ng/mL) Fig. 4. Bioactivity assay of rhSCF on human TF1 cells by ATP assay kit. Comparison of cell viability of TF1 cells incubated with purified rhSCF (d, solid line) and commercial rhSCF (j, dotted line).

purification system, a quickly and highly purified protein product can be obtained that lacks any extra amino acid residues derived from the Profinity eXact™ tag. At first, fusion protein of eXact-hSCF did not bind to resin in standard protocol using sodium phosphate buffer. Generally, in this case, the tag may otherwise tend to be buried in the inner side of the oligomeric protein and thus inaccessible to the affinity resin. According to previous reports, SCF forms the dimer in solution [12] and this structure in solution may prevent the binding between N-terminal tag and resin. In His-tag fusion protein purification system, some denaturants (8 M urea, 6 M guanidine-HCl or others) can be used to expose the His-tag completely, whereas in the Profinity resin immobilized mutant subtilisin protein system, it is not acceptable to use strong denaturants in chromatography. Another possibility could be the masking of the tag by protein–endotoxin interactions or protein aggregation. Thus we tried to dissociate the concomitant endotoxin from the protein surface by using two methods, i.e., aqueous two-phase micellar systems using non-ionic detergent Triton X-114 and binding buffer containing 0.5 M L-arginine. These two treatments dramatically improved the binding and recovery in affinity chromatography. Especially, L-arginine was more effective than Triton X-114 treatment for improvement of chromatography performance in yield and purity. L-Arginine as chaotropic cation may prevent protein aggregation or oligomer formation of rhSCF,

A

resulting in exposing the eXact tag domain and dramatically improving binding to the resin. The values of dissociation rate constant (Kd) of eXact-tag and Profinity-resin is 100 pM [28] and Kd of rhSCF dimer is 3.7 nM [41], respectively. Owing to this difference of Kd value, fusion protein may preferentially form a monomer than a dimer under L-arginine condition, resulting in efficient binding to the resin. Some groups have shown that L-arginine is effective for improving column chromatographic performances [31], e.g., dissociation of protein and reduction of endotoxin from Protein A-bound antibodies [32–34], reduction of non-specific binding of proteins in gel permeation chromatography [35], and improvement of elution recovery in hydrophobic chromatography and dye-affinity chromatography [36,37]. Here we demonstrated that L-arginine greatly enhances the binding and elution recovery of bound hSCF from Profinity eXact™ affinity chromatography. L-Arginine is mild reagent for both this affinity resin and recombinant protein compared to other chaotropic agent (urea and guanidine) and detergents (SDS, TritonX, etc). As demonstrated, the purified hSCF had a dose-dependent positive effect on human TF-1 viability, preventing cell death via c-Kit [39]. Our data consistently indicated that recombinant hSCF exhibits properties comparable to those reported for the native protein. We also describe the functional tests using human mast cell line LAD2. Red granules stained with Safranin were increased in LAD2 cells cultured with SCF (100 ng/ml) compared to those without SCF. This result shows that LAD2 cells cultured with SCF were at the stage of maturation into the connective tissue type mast cells by heparin production. Thus, the purified hSCF according to our novel methodology is shown to be capable of inducing differentiation on LAD2 cells. Given recent advances in stem cell research, there is a growing demand for hSCF as medium supplement. Techniques to generate human mast cells from peripheral CD 34+ stem cells [42] and human embryonic stem (ES) cells [43] have been developed with a combination of cytokines including rhSCF. Hematopoiesis from human ES cells been shown by stroma cell co-culture system using rhSCF combined with several cytokines [17]. Also hematopoietic stem cell from human induced pluripotent stem (iPS) cells was generated by teratoma coinjected with hSCF [18]. Furthermore, the induction of functional mature neutrophils from human iPS cells was reported using rhSCF combined with murine feeder cell co-culture system [44]. These reports opened the door for a new era of human stem cell research and regenerative medicine. We believe that the development of stem cell research will enhance the demand for hSCF. Therefore our present work could contribute to this basic research.

B

Fig. 5. Effects of rhSCF protein on growth and differentiation on human mast cell LAD2. Alcian-blue safranin staining of untreated (A) and SCF-treated (B) human LAD2 cells (400). Scale bars: 10 lm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

T. Akuta et al. / Protein Expression and Purification 105 (2015) 1–7

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