Appl Biochem Biotechnol (2008) 144:181–189
`DOI 10.1007/s12010-007-8112-0
`
`Solubilization and Refolding with Simultaneous
`Purification of Recombinant Human Stem Cell Factor
`
`Chaozhan Wang & Jiahua Liu & Lili Wang & Xindu Geng
`
`Received: 14 June 2007 / Accepted: 21 November 2007 /
`Published online: 5 January 2008
`# Humana Press Inc. 2008
`
`Abstract Recombinant human stem cell factor (rhSCF) was solubilized and renatured from
`inclusion bodies expressed in Escherichia coli. The effect of both pH and urea on the
`solubilization of rhSCF inclusion bodies was investigated; the results indicate that the sol-
`ubilization of rhSCF inclusion bodies was significantly influenced by the pH of the solution
`employed, and low concentration of urea can drastically improve the solubilization of rhSCF
`when solubilized by high pH solution. The solubilized rhSCF can be easily refolded with
`simultaneous purification by ion exchange chromatography (IEC), with a specific activity
`−1, a purity of 96.3%, and a mass recovery of 43.0%. The presented
`of 7.8×105 IU·mg
`experimental results show that rhSCF solubilized by high pH solution containing low
`concentration of urea is easier to be renatured than that solubilized by high concentration of
`urea, and the IEC refolding method was more efficient than dilution refolding and dialysis
`refolding for rhSCF. It may have a great potential for large-scale production of rhSCF.
`
`Keywords Recombinant human stem cell factor . Solubilization of inclusion bodies .
`Protein refolding . Purification . Ion exchange chromatography . Protein folding
`liquid chromatography
`
`Introduction
`
`Stem cell factor (SCF, also called steel factor or c-kit ligand) is a multipotent hematopoietic
`growth factor for early progenitor cells of different lineages [1, 2]. Stem cell factor can act
`on hematopoiesis by stimulating the survival and proliferation of stem cells and progenitor
`cells. It is also crucial for mast cell production and function and plays an important role in
`the development of melanocytes, germ cells, and intestinal pacemaker cells [1]. SCF exists
`naturally as membrane-anchored and soluble isoforms as a result of alternative RNA
`splicing and proteolytic processing [3]. Each SCF monomer contains two intra-chain
`
`C. Wang (*) : J. Liu : L. Wang : X. Geng
`Institute of Modern Separation Science, Department of Chemistry, Northwest University,
`229 Tai Bai North Road, Xi’an 710069, People’s Republic of China
`e-mail: czwang@nwu.edu.cn
`
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`disulfide bridges, Cys4-Cys89 and Cys43-Cys138, as well as three potential N-linked sites of
`glycosylation, Asn65, Asn72, and Asn120. The presence or absence of glycosylation does not
`affect its specific activity [4]. Potential therapeutic applications of SCF in clinic trials
`include the treatment of anemia, boosting the mobilization of hematopoietic stem/
`progenitor cells to the peripheral blood for harvest and transplantation, and increasing the
`effectiveness of gene therapy [1, 5].
`Recombinant human SCF (rhSCF) has been expressed in Escherichia coli by many
`laboratories including ours [6]. But rhSCF protein often forms insoluble and inactive
`inclusion bodies in E. coli. A general strategy for recovery of active rhSCF from inclusion
`bodies involves cell lysis, extraction and cleaning of inclusion bodies, solubilization of
`inclusion bodies, and refolding into its native conformation [7, 8]. rhSCF inclusion bodies
`−1 urea or
`were usually solubilized by high concentration of denaturants, such as 8.0 mol·l
`−1 guanidine hydrochloride (GuHCl), reducing agents, such as dithiothreitol or β-
`7.0 mol·l
`mercaptoethanol (β-ME), are added to reduce all disulfide bonds. Then, the denatured
`protein is transferred into a nondenaturating environment to shift the folding equilibrium
`toward its native conformation. This is normally achieved by removing the denaturants
`through dilution or dialysis in the presence of reduced glutathione (GSH) and oxidized
`glutathione (GSSG). However, refolding yields are typically low. Low refolding yields are
`attributed to mass loss of protein by aggregation because of nonspecific hydrophobic
`interactions. Dilution of the solubilized/denatured protein significantly increases sample
`volume, bring difficulty to subsequent chromatographic purification process, and increase
`costs. Therefore, to develop a new protocol to recover active rhSCF from inclusion bodies
`is very necessary for the production of rhSCF from E. coli.
`It was reported that high concentrations of urea or guanidine hydrochloride (GuHCl),
`being strong denaturants, result in the loss of existing native-like secondary structures of the
`target protein in the inclusion bodies [9] and lead to easy aggregation during protein
`refolding. In recent years, high pH solution has been used to solubilize proteins in inclusion
`bodies expressed in E. coli [10–12], and the results showed that this solubilization method
`is beneficial to protein refolding. Recently, liquid chromatography (LC) has been used to
`refold proteins with higher yields [13–17]. The main advantage of the LC refolding method
`is that it not only prevents the unfolded protein molecules from aggregating with each other
`but also simultaneously purifies or partially purifies the protein during the chromatographic
`process; thus, it is called protein folding liquid chromatography (PFLC) [13, 18]. Ion
`exchange chromatography (IEC) is a widely used chromatographic method for protein
`purification; it was reported that about 70% of protocol for protein purification involved
`IEC, and now, IEC has been becoming one of the most frequently used LC refolding
`methods and has been applied to many proteins with high yields [19–28].
`In the presented work, high pH buffers were used to solubilize rhSCF expressed in E.
`coli as inclusion bodies; the high pH buffer component and the solubilization conditions
`were optimized, then the solubilized rhSCF was refolded by dilution, dialysis, and IEC,
`respectively, and the refolding results were compared with the urea solubilized rhSCF.
`
`Experimental
`
`Instruments
`
`LC-10A high-performance liquid chromatograph (Shimadzu, Japan), consisting of two LC-
`10ATVP pumps, one SPD-10AVP UV-Vis detector, one SCL-10AVP controller, and one
`
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`Appl Biochem Biotechnol (2008) 144:181–189
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`183
`
`Rheodyne 7725 injection valve. All chromatographic data was collected and evaluated
`using the class-VP data system. Strong anion exchange chromatographic packings were
`prepared in our laboratory and packed into a column (10×1.2 cm I.D.). The electrophoresis
`apparatus were obtained from Amersham Pharmacia Biotech (Uppsala, Sweden). An
`AvantiTM J-25 centrifuge (Beckman coulterTM, USA) was used for centrifugation. A 5 l
`fermentor (Braun, Germany) was used to express protein.
`
`Chemicals
`
`Acrylamide and bis-acrylamide, GSH, and GSSG are of analytical grade, obtained from
`Sigma (USA). Tris, glycine, and sodium dodecyl sulfate (SDS) were obtained from
`Amersco (USA). Bovine serum albumin (BSA) was from Sigma Chemicals (USA).
`Molecular mass marker was obtained from Amersham Pharmacia Biotech (Uppsala,
`Sweden). All other chemicals were of analytical grade.
`
`Expression of rhSCF
`
`A fed-batch fermentation was carried out in a 5-l bioreactor with a working volume of 4 l,
`−1 glycerol, 5 g·l
`
`−1 tryptone, 5 g·l−1 yeast extract, and M9 salts. Fermentation
`with 10 g·l
`−1 NaOH
`was performed at 32 °C, and the pH of medium was maintained at 7.2 by 5 mol·l
`with the dissolved O2 concentration held at 30%. When the culture reached an OD600 of 4,
`the temperature was shifted to 42 °C to induce rhSCF synthesis. The culture was harvested
`−1). The bacteria were harvested and resuspended in
`at an OD600 of 7.8 (= cell dry wt 5.6 g·l
`−1 NaH2PO4/NaOH, pH 7.4 by centrifugation for 10 min at 25,000×g, 4 °C.
`a 0.05 mol·l
`
`Recovery of rhSCF Inclusion Bodies
`
`−1 Tris–HCl
`The cells were thawed at room temperature and cleaned up with 0.020 mol·l
`(pH 8.0), and then, the suspension was centrifuged at 7,000 rpm and 4 °C for 10 min after
`washing. The supernatant was discarded. After freezing at −20 °C for 12 h, 100 g of the
`−1
`frozen cells were thawed at room temperature and resuspended in 1,000 ml of 0.050 mol·l
`−1 ethylenediaminetetraacetic acid (EDTA).
`Tris–HCl buffer (pH 8.0) containing 1.0 m mol·l
`The cells were lysed by sonication on ice-water bath. The lysates were centrifuged at
`14,000 rpm for 20 min to collect the insoluble protein aggregates. The pellet (protein
`aggregates and cell debris) was washed with 500 ml of the following solutions:
`−1 Tris–HCl (pH 8.0) containing 0.010 mol·l
`−1 EDTA and 2.0 mmol·l
`−1 β-
`0.020 mol·l
`−1 Tris–HCl (pH 8.0) containing 2.0 mol·l
`−1 urea and 2.0 m mol·l
`−1 EDTA,
`ME, 0.020 mol·l
`−1 Tris–HCl (pH 8.0) containing 70% 2-propanol, respectively. Finally, the
`and 0.020 mol·l
`−1 Tris–HCl (pH 8.0). After each washing
`inclusion bodies were washed with 0.02 mol·l
`step, the suspension was centrifuged at 14,000 rpm and 4°C for 15 min, the supernatant was
`discarded. Then, the pellet fraction containing rhSCF inclusion bodies were obtained and
`stored at −20 °C.
`
`Solubilization of rhSCF from Inclusion Bodies
`
`Several batches of 1.0 g of purified rhSCF inclusion bodies were solubilized in 20 ml of
`−1 Tris containing 0.05 mol·l
`−1 Na2HPO4 with different pH adjusted
`solution I (0.05 mol·l
`−1 Tris, pH 12.5
`by hydrochloride acid or sodium hydroxide), solution II (0.05 mol·l
`−1 urea), solution III (8.0 mol·l
`−1 Na2HPO4 and 2.0 mol·l
`−1 urea
`containing 0.05 mol·l
`
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`
`−1 β-
`−1 EDTA; and 0.1 mol·l
`−1Tris, pH 8.0; 0.02 mol·l
`containing 0.1 mol·l
`mercaptolethanol). For solubilization by solutions I and II, the rhSCF suspension was
`−1 HCl to pH 10.0 and was continuously stirred for 2 h; after
`adjusted by using 0.1 mmol·l
`−1 HCl, then the suspension was
`that, the pH was adjusted to 8.0 by using 0.1 mmol·l
`centrifuged at 14,000 rpm for 20 min to remove insoluble debris, and the supernatant was
`kept at 4 °C for renaturation and purification. For solubilization by solution III, the rhSCF
`inclusion bodies was solubilized with continuous stirring for 4 h, then the suspension was
`centrifuged at 14,000 rpm for 20 min, and the supernatant containing rhSCF was collected
`for further use.
`
`Procedures for the Refolding with Simultaneous Purification of rhSCF by IEC
`
`Chromatographic runs were carried out at room temperature using a strong anion exchange
`column (10× 1.2 cm I.D.) and connected to a LC-10A high-performance liquid
`−1
`chromatograph. The column was equilibrated with solution A consisting of 1 mmol·l
`−1 Tris (pH 8.0), 1.0 mmol·l
`−1 GSH, and 0.1 mmol·l
`−1 GSSG. Four
`EDTA, 20 mmol·l
`hundred microliters of sample solution containing the solubilized and denatured rhSCF was
`directly injected into the column. After washing the column with 10 ml of the solution A,
`the refolding with simultaneous purification of rhSCF was accomplished after a linear
`gradient elution from 100% A to 100% B (solution B consisted of solution A plus
`−1 NaCl) in 30 min with a delay of 10 min at a flow rate of 2.0 ml·min
`−1. The
`1.0 mol·l
`profile was recorded with a UV detection at 280 nm.
`
`Refolding of rhSCF by Dilution
`
`Four hundred microliters of sample solution containing the denatured rhSCF was diluted
`
`
`−1 Tris (pH 8.0), 1 mmol·l−1 EDTA, 1.0 mmol·l−1 GSH,
`100-fold with 20 mmol·l
`−1 GSSG, then the solution was left for 24 h at 4 °C. After refolding, the
`0.1 mmol·l
`rhSCF was purified by IEC.
`
`Refolding of rhSCF by Dialysis
`
`−1
`−1 Tris (pH 8.0), 1 mmol·l
`The, denatured rhSCF solution was dialyzed against 20 mmol·l
`
`EDTA, 1.0 mmol·l−1 GSH, 0.1 mmol·l
`−1 GSSG at 4 °C with continuous stirring for 48 h;
`the buffer was renewed each 4 h during the dialysis. After refolding, the rhSCF was purified
`by IEC.
`
`Analytical Procedures
`
`Electrophoresis
`
`SDS–polyacrylamide gel electrophoresis (SDS–PAGE) using a Tris–SDS–glycine buffer
`system in the presence of a reducing agent was used to detect the purity of the purified
`rhSCF contained in the fractions after IEC. Electrophoresis was performed for 1 h at 250 V
`using 15% polyacrylamide gels.
`
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`185
`
`Determination of Protein Concentration and Mass Recovery
`
`The protein concentration was estimated by Bradford quantitative protein determination
`assay using BSA as standard. The mass recovery (Rm) of rhSCF was defined as
`
`ð1Þ
`mG;IB ¼ CF VF PFð
`Þ= CIB VIB PIBð
`Þ
`Rm ¼ mG;F
`
`
`where, mG,F, the mass of rhSCF in the finally obtained rhSCF solution (mg); CF, total
`−1); VF, volume of the
`protein concentration in the finally obtained rhSCF solution (mg·ml
`finally obtained rhSCF solution (ml); PF, purity of rhSCF in the finally obtained rhSCF
`solution; mG,IB, the mass of rhSCF in the injected solution of inclusion bodies (mg); CIB,
`−1); VIB,
`total protein concentration in the injected solution of inclusion bodies (mg·ml
`volume of the injected solution of inclusion bodies (ml); and PIB, purity of rhSCF in the
`injected solution of inclusion bodies.
`
`Bioactivity Assay of rhSCF
`
`The bioassay for the bioactivity of the renatured rhSCF was determined using a UT-
`7-dependent cell line as described previously [29].
`
`Results and Discussion
`
`Effect of pH on the Solubilization of rhSCF Inclusion Bodies
`
`It was previously shown that proteins in inclusion bodies expressed in E. coli can be
`solubilized by high pH solution [10–12], the obtained denatured proteins is easy to be
`renatured with relatively high yields. In the presented work, the same amount of rhSCF
`−1
`−1 Tris buffer containing 0.05 mol·l
`inclusion bodies were solubilized by 0.05 mol·l
`Na2HPO4 with pH from 8.0 to 13.5, respectively, and the solubilization was monitored by
`determining protein concentration using UV absorbance at 280 nm and by determining
`
`Absorbance at 450 nm
`
`0.5
`
`0.4
`
`0.3
`
`0.2
`
`00
`
`.1
`
`0.6
`
`0.5
`
`0.4
`
`0.3
`
`0.2
`
`0.1
`
`Absorbance at 280 nm
`
`Fig. 1 Solubilization of rhSCF at
`different pH. One gram of rhSCF
`inclusion bodies was solubilized
`in 20 ml of 0.05 mol·l−1 Tris
`−1
`buffer containing 0.05 mol·l
`Na2HPO4 at pH from 8.0 to 13.5
`
`0
`7.5
`
`8.5
`
`9.5
`
`11.5
`10.5
`pH
`
`12.5
`
`13.5
`
`A280nm
`
`A450nm
`
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`
`2
`4
`6
`Urea concentration (mol˙l-1)
`
`8
`
`10
`
`0.6
`
`0.5
`
`0.4
`
`0.3
`
`0.2
`
`0.1
`
`0
`
`Absorbance at 280 nm
`
`Fig. 2 Effect of urea concentra-
`tion on the solubility of rhSCF
`inclusion bodies. One gram of
`rhSCF inclusion bodies was
`solubilized in 20 ml of
`0.05 mol·l−1 Tris containing
`−1 Na2HPO4 at pH 13.0
`0.05 mol·l
`−1 urea
`and 0~8.0 mol·l
`
`turbidity using visible absorbance at 450 nm. The results were shown in Fig. 1. It can be
`seen from this figure that solubilization of rhSCF inclusion bodies was very poor and
`hardly influenced by the pH in the pH range from 8.0 to 11.5. Remarkable enhancement of
`solubilization was observed with further increasing of pH, with a maximum at pH 13.0.
`
`Effect of Urea on the Solubilization of rhSCF Inclusion Bodies
`
`Urea is a widely used solubilizing agent for inclusion bodies, and it was also usually used to
`solubilize rhSCF inclusion bodies. From the above experimental results, rhSCF inclusion
`bodies can be solubilized with a high pH buffer. However, what is the result when high pH
`and urea were combined to solubilize rhSCF inclusion bodies? Figure 2 shows the
`solubilization effect of high pH buffer containing different concentration of urea. The
`results show that the solubilization of rhSCF inclusion bodies was greatly increased by
`−1
`−1 Tris buffer containing 0.05 mol·l
`introducing low concentration of urea in 0.05 mol·l
`Na2HPO4 at pH 13.0; an approximate plateau was approached when the urea concentration
`−1.
`was enhanced to 2.0 mol·l
`
`Refolding with Simultaneous Purification of the High pH Solubilized rhSCF by IEC
`
`Liquid chromatography has been recently applied to protein refolding; its main advantages
`are that proteins can be purified simultaneously during protein refolding; refolding yields
`are relatively high. IEC is a most commonly used LC method for protein refolding. Here,
`
`10
`
`t/min
`
`20
`
`0.10
`
`0.05
`
`Absorbance(280nm)
`
`Fig. 3 Chromatogram of
`rhSCF refolded by IEC.
`Chromatographic conditions:
`gradient/linear gradient from 0%
`B to 100% B in 30 min, with a
`delay of 10 min; flow rate is
`2.0 ml·min−1; mobile phase A:
`0.05 mol·l−1 Tris (pH = 8.0),
`−1 EDTA, 1.0 mmol·l
`1 mmol·l
`GSH, 0.1 mmol·l−1 GSSG;
`mobile phase B: the mobile phase
`A containing 1 mol·l−1 NaCl; the
`asterisk denotes rhSCF
`
`−1
`
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`187
`
`Fig. 4 SDS–PAGE analysis
`of rhSCF. 1 rhSCF refolded by
`IEC with simultaneous
`purification; 2 extract of rhSCF
`inclusion bodies by high pH
`buffer containing 2.0 mol·l−1 urea
`
`−1 urea;
`IEC was used to refold rhSCF solubilized by high pH buffer containing 2.0 mol·l
`the chromatogram is shown in Fig. 3. The whole IEC refolding process could be
`accomplished in 1 h, including the equilibration and elution program. The obtained rhSCF
`−1, a mass recovery (MR) of 43.0%, and a
`has a specific bioactivity (SB) of 7.8×105 IU·mg
`purity of 96.3% (Fig. 4). For comparison,
`the rhSCF solubilized by high pH buffer
`−1 urea was also refolded by dilution and dialysis, and the above used
`containing 2.0 mol·l
`−1
`three refolding methods were also applied to refold the rhSCF solubilized by 8.0 mol·l
`urea. The results are shown in Table 1. It can be seen from this table that all of the mass
`
`Table 1 Comparison of results for rhSCF solubilized and refolded by using a different method.
`
`rhSCF
`sample
`
`SBdilution
`(IU·mg−1)
`
`MRdilution
`(%)
`
`SBdialysis
`(IU·mg−1)
`
`MRdialysis
`(%)
`
`SBIEC
`(IU·mg−1)
`
`MRIEC
`(%)
`
`a
`
`(3.3±0.94)×105
`(4.5±1.1) ×105
`18.8±1.53
`rhSCFurea
`b
`(4.7±0.86) ×105
`(5.4±1.4) ×105
`25.4±2.16
`rhSCFpH
`a rhSCFurea presents the rhSCF solubilized by 8.0 mol·l−1 urea.
`b rhSCFpH presents the rhSCF solubilized by 0.05 mol·l-1 Tris (pH 13.0) containing 0.05 mol·l
`and 2.0 mol·l−1 urea.
`
`16.8±1.07
`26.2±1.79
`
`(7.6±1.8) ×105
`(7.8±1.5) ×105
`
`36.4±3.10
`43.0±2.93
`
`−1 Na2HPO4
`
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`−1 urea are higher
`recovery for rhSCF solubilized by high pH buffer containing 2.0 mol·l
`−1 urea, no matter which refolding method was employed,
`than that solubilized by 8.0 mol·l
`and their specific bioactivities were comparable. This may attribute to that rhSCF
`solubilized from the inclusion bodies without disturbing its existing native-like secondary
`structure in the high pH buffer [9, 30], which helped in lowering the extent of protein
`aggregation during rhSCF refolding. It can also be seen from Table 1 that both of the SB
`and MR of the rhSCF refolded by IEC are higher than those refolded by dilution or dialysis,
`no matter which solubilizing method was used.
`In the previous literature [4], rhSCF expressed in E. coli as inclusion bodies was
`−1 urea solution, refolded and oxidized by dilution refolding with a
`solubilized by 8 mol·l
`buffer containing low concentration of urea and glutathione for 60 h; the renatured rhSCF
`solution was concentrated by ultrafiltration and buffer exchanged by using diafilter, then the
`crude protein solution was primarily purified by acid precipitation, and filtration was used
`to remove the precipitated contaminant. After that, several chromatographic steps were used
`to further purify the rhSCF. Firstly, strong cation exchange chromatography was applied,
`then reversed-phase chromatography, strong anion exchange chromatography, and size
`exclusion chromatography were followed in sequence. The final yield of rhSCF was only
`18%. In the present work, rhSCF inclusion bodies were solubilized by high-pH buffer with
`a low concentration of urea, and the solubilized rhSCF was refolded by strong anion
`exchange chromatography. As a result, rhSCF was also purified during the refolding
`process without further treatment, and a mass recovery of 43.0% was obtained; it was much
`higher than that in the early work [4].
`
`Conclusions
`
`The effect of pH and urea on the solubilization of rhSCF inclusion bodies was investigated;
`the results indicate that the solubilization of rhSCF inclusion bodies was significantly
`influenced by the pH of the solution employed, and low concentration of urea can
`drastically improve the solubilization of rhSCF using high pH solution. The solubilized
`rhSCF can be easily refolded with simultaneous purification by IEC with relatively high
`efficiency. The rhSCF solubilized by high pH solution containing low concentration of urea
`is easier to be refolded than that solubilized by high concentration of urea, and the IEC
`refolding method was more efficient than dilution refolding and dialysis refolding for
`rhSCF. The reported solubilization and refolding method may also be useful for other
`proteins produced in E. coli as inclusion bodies.
`
`Acknowledgment This work was supported by the National Natural Science Foundation in China (no.
`20705028) and the Foundation of Key Laboratory of Modern Separation Science in Shaanxi Province
`(no. 05JS61).
`
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