Applied
`i <ffirehemistry
`and Biotechnology
`
`,
`
`I
`
`Editor-In -Chief Ashok Mulchandani
`
`* HUMANA PRESS
`
`1 of 11
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`Applied Biochemistry and Bi~technology
`Part A: Enzyme Engineering and Biotechnology
`
`·
`
`Editorial Board
`
`Ashok Mulchandani • Editor-In-Chief
`Department of Chemical and
`Environmental Engineering
`Bourns Hall, Room A242
`University of California
`Riverside, CA 92521
`E-mail: adani@engr.ucr.edu
`Advisory Board
`Howard H. Weetall • Founding Editor
`US Environmental Protection Agency • Las Vegas, NV
`David R. Walt • Former Editor-In-Chief
`Department of Chemistry • Tufts University• Medford, MA
`Isao Karube
`Research Center/or Advanced Science and Technology•
`University of Tokyo• Tokyo 153, Japan
`Klaus Mosbach
`Department of Pure and Applied Biochemistry•
`University of Lund • Lund, Sweden
`Shuichi Suzuki
`Saitama Institute o/Teclmology • Saitama, Japan
`Associate Editors
`Wilfred Chen
`Depart me Ill of Chemical and Enviromnemal Engineering •
`University of California• Riverside, CA
`Elisabeth Csoregi
`Department of Biotechology • University of Lund• Lund, Sweden
`David W. Murhammer
`Departme/11 of Chemical and Biochemical Engineering •
`University of Iowa • Iowa City, IA
`Anup K. Singh
`Biosystems Research Department • Sandia National Laboratories •
`Livermore, CA
`
`M. Aizawa, Tokyo lnstitllfe o/Teclmology, Tokyo, Japan
`M.A. Arnold, University of Iowa, Iowa Cif)\ IA
`L. Bachas, University of KentuckJ\ Lexington, KY
`T. T. Bachmann, Universif)• o/Stllttgartt, Swttgart, Germany
`S. Belkin, The Hebrew Universif)• of Jerusalem, Jerusalem, Israel
`Harvey W. Blanch, Universif)• of California, Berkeley, CA
`H.J. Cha, Pohang Universif)• of Science and Technology, Pohang, Korea
`Q. Chuan-Ling, lnstitllfe of Z.OologJ\ Chinese Academy of Sciences, Beijing, China
`Nancy A. Da Silva, University of California, Irvine, CA
`M. DeLisa, Cornell University, Ithaca, NY
`M. Deshusses, University o/Califomia, Riverside, CA
`J. S. Dordick, Rensselaer Polytechnic Institute, Troy, NY
`M. E. Eldefrawi, University of Maryland, Baltimore, MD
`M. B. Gu, K-JIST, Gwangju, Korea
`R. K. Jain, Institute of Microbial Technology, Chandigarh, India
`N. G. Karanth, Ce111ral Food and Technology Research Institllfe, Mysore, India
`R. Kelly, North Carolina State University, Raleigh, NC
`A. M. Klibanov, M.I. T., Cambridge, MA
`U. J. Krull, Erindale College, Universif)• o/Torollfo, Mississauga, O111ario, Canada
`M. R. Ladish, Purdue Universif)•, West Lafayette, IN
`K. Lee, Come/I Universif)•, Ithaca, NY
`Y. Y. Lee, Auburn Universif)1 Auburn AL
`F. S. Ligler, Naval Research Laborat01y, Washington, DC
`R. Linhardt, Universif)• of Iowa, Iowa Cit.)\ IA
`A. Pandey, Regional Research Laboratory, Trivandrum, India
`M. Pishko, The Pennsylvania State Universif)•, University Park, PA
`V. Renugopalakrishnan, Harvard Medical School, National Universif)' of
`Singapore
`D. Ryu, Universif)• of Califomia, Davis, CA
`M. Seibert, National Renewable Energy Laborato,y, Golden, CO
`W. Tan, Uni1•ersity of Florida, Gainsville, Fl
`Mitsuyoshi Ueda, Kyoto University, Kyoto, Japan
`S. D. Varfolomeyev, M. V. lomonosov Moscow State University, Moscow, Russia
`J.-H. Xu, East China Universif)• of Science and Technology, Shanghai, China
`P. Wang, University of Akron, Akron, OH
`C. E. Wyman, University o/Califomia, Riverside, Riverside, CA
`H. Zhao, Univerisf)• of Jllinois, Urbana Champagne, IL
`Patents and Literature Reviews Editor:
`MarkR. Riley
`Dept. of Agrirnltura/ & Biosystems £11gi11eerir,g • Shantz Bldg.
`Unfrersit)' of Arizona• Tucson, AZ 85721-0338
`Reviews in Biotechnology Editor:
`John M. Walker
`University of Henfordshire • Hatfield • Herts • UK
`Volume 144, Number 2, February 2008
`Copyright© 2008 Humana Press Inc. All Rights Reserved.
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`ISSN 0273-2289 (Print)/1559-0291 (Online)
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`Applied Bioche111ist1ya11d Biotechnology is made available for abstracting or indexing in Chemical Abstracts, Biological Abstracts, Curre11t Co111ents, Scie11ce
`Citation Index, EMBASF/Exce,pta Medica, Index Medicus, Cambridge Scie11tific Abstracts, Reference Update, and related compendia.
`
`Assistant Editor
`Priti Mulchandani
`Department of Chemical and Enviro11111e11tal Engineering •
`University of California • Riverside, CA
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`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(cid:173)
`ubilization ofrhSCF inclusion bodies was significantly influenced by the pH of the solution
`employed, and low concentration ofurea can drastically improve the solubilization ofrhSCF
`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
`of 7.8x 105 IU·mg- 1
`, a purity of 96.3%, and a mass recovery of 43.0%. The presented
`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 [l, 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 (Bi) · J. Liu · L. Wang · X. Geng
`Institute of Modem 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-Cys 138
`, as well as three potential N-linked sites of
`, and Asn 120
`glycosylation, Asn65
`, Asn72
`. The presence or absence ofglycosylation 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 [I, 5).
`Recombinant human SCF (rhSCF) has been expressed in Escherichia coli by many
`laborat01ies 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
`were usually solubilized by high concentration of denaturants, such as 8.0 mol·l- 1 urea or
`7.0 molr 1 guanidine hydrochloride (GuHCI), reducing agents, such as dithiothreitol or/)(cid:173)
`mercaptoethanol (/)-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 (GuHCI),
`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 £ .
`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-IOA high-performance liquid chromatograph (Shimadzu, Japan), consisting of two LC(cid:173)
`I0ATVP pumps, one SPD-I0AVP UV-Vis detector, one SCL-I0AVP controller, and one
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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 (!Ox 1.2 cm I.D.). The electrophoresis
`apparatus were obtained from Amersham Phannacia Biotech (Uppsala, Sweden). An
`Avanti™ J-25 centrifuge (Beckman coulterTM, USA) was used for centrifugation. A 5 I
`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 (SOS) 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 fennentation was carried out in a 5-1 bioreactor with a working volume of 4 I,
`with 10 g·l- 1 glycerol, 5 g·l- 1 tryptone, 5 g-1- 1 yeast extract, and M9 salts. Fermentation
`was performed at 32 °C, and the pH of medium was maintained at 7.2 by 5 mol·l- 1 NaOH
`with the dissolved 0 2 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
`at an OD600 of7.8 (= cell dry wt 5.6 g-1- 1
`). The bacteria were harvested and resuspended in
`a 0.05 mol·l- 1 NaH2POJNaOH, pH 7.4 by centrifugation for 10 min at 25,000xg, 4 °C.
`
`Recovery of rhSCF Inclusion Bodies
`
`The cells were thawed at room temperature and cleaned up with 0.020 molT 1 Tris- HCI
`(pH 8.0), and then, the suspension was centrifuged at 7,000 rpm and 4 °C for IO min after
`washing. The supernatant was discarded. After freezing at -20 °C for 12 h, 100 g of the
`frozen cells were thawed at room temperature and resuspended in 1,000 ml of0.050 mol·l- 1
`Tris-HCI buffer (pH 8.0) containing 1.0 m mol·l- 1 ethylenediaminetetraacetic acid (EDTA).
`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:
`0.020 motr 1 Tris- HCI (pH 8.0) containing 0.010 mol-1- 1 EDTA and 2.0 mmol·l- 1 /3-
`ME, 0.020 molT 1 Tris-HCI (pH 8.0) containing 2.0 mol-1- 1 urea and 2.0 m mol·l- 1 EDTA,
`and 0.020 mol·l- 1 Tris-HCI (pH 8.0) containing 70% 2-propanol, respectively. Finally, the
`inclusion bodies were washed with 0.02 molT 1 Tris-HCI (pH 8.0). After each washing
`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
`solution I (0.05 mol·l- 1 Tris containing 0.05 molT 1 Na2HPO4 with different pH adjusted
`by hydrochloride acid or sodium hydroxide), solution II (0.05 mol·l- 1 Tris, pH 12.5
`containing 0.05 molT 1 Na2HPO4 and 2.0 mol·l- 1 urea), solution III (8.0 mol-1- 1 urea
`
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`containing 0.1 mo1T 1Tris, pH 8.0; 0.02 mol·l- 1 EDTA; and 0.1 molT 1
`/)(cid:173)
`mercaptolethanol). For solubilization by solutions I and II, the rhSCF suspension was
`adjusted by using 0.1 mmol-1- 1 HCI to pH 10.0 and was continuously stirred for 2 h; after
`that, the pH was adjusted to 8.0 by using 0.1 mmol-1- 1 HCl, then the suspension was
`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 (lOx 1.2 cm I.D.) and connected to a LC- lOA high-performance liquid
`chromatograph. The column was equilibrated with solution A consisting of I mmol-1- 1
`EDTA, 20 mmo1T 1 Tris (pH 8.0), 1.0 mmol-1- 1 GSH, and 0.1 mmol-1- 1 GSSG. Four
`hundred microliters of sample solution containing the solubilized and denatured rhSCF was
`directly injected into the column. After washing the column with l 0 ml of the solution A,
`the refolding with simultaneous purification of rhSCF was accomplished after a linear
`gradient elution from i00% A to 100% B (solution B consisted of solution A plus
`1.0 mo1T1 NaCl) in 30 min with a delay of 10 min at a flow rate of 2.0 ml·min- 1
`• The
`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
`100-fold with 20 mmo1T 1 Tris (pH 8.0), I mmol·l- 1 EDTA, 1.0 mmol-1- 1 GSH,
`0.1 mmolT 1 GSSG, then the solution was left for 24 h at 4 °C. After refolding, the
`rhSCF was purified by IEC.
`
`Refolding of rhSCF by Dialysis
`
`The, denatured rhSCF solution was dialyzed against 20 mmo1T 1 Tris (pH 8.0), I mmol-1- 1
`EDTA, 1.0 mmol-1- 1 GSH, 0.1 mmolT1 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 l 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
`
`(1)
`where, ma.F, the mass of rhSCF in the finally obtained rhSCF solution (mg); CF, total
`protein concentration in the finally obtained rhSCF solution (mg·ml- 1
`); VF, volume of the
`finally obtained rhSCF solution (ml); PF, purity of rhSCF in the finally obtained rhSCF
`solution; ma,rn, the mass of rhSCF in the injected solution of inclusion bodies (mg); C18,
`total protein concentration in the injected solution of inclusion bodies (mg·ml- 1
`); Vis,
`volume of the injected solution of inclusion bodies (ml); and P18, 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
`inclusion bodies were solubilized by 0.05 mol-1- 1 Tris buffer containing 0.05 mol-1- 1
`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
`Fig. I Solubilization ofrhSCF at
`0.6
`different pH. One gram of rhSCF
`inclusion bodies was solubilized
`in 20 ml of 0.05 molr' Tris
`buffer containing 0.05 moir'
`Na2HPO4 at pH from 8.0 to 13.5
`
`0.5
`
`►
`0.4 0-
`"' 0 ..,
`
`0.3
`
`0-
`f>)
`;:l
`(')
`(1)
`
`0.5
`
`0.4
`
`0.3
`
`► 0-
`"' 0 ..,
`0-
`f>)
`;:l
`(')
`(1)
`~
`Iv
`00
`0
`;:l
`3
`
`0.2
`
`0.1
`
`0
`7.5
`
`8.5
`
`9.5
`
`10.5 11.5 12.5 13.5
`pH
`
`-A280nm _..._ A450nm
`
`0.2 ~
`.j:>.
`Vl
`0
`;:l
`3
`
`0.1
`
`0
`
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`Appl Biochem Biotechnol (2008) 144: 181- 189
`
`0.6
`
`V,
`
`0.2
`
`►
`cr' 0.5
`0 ....
`cr'
`"" 0.4
`::,
`(')
`0
`~ 0.3
`N
`00
`0
`::,
`3
`
`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 molr 1 Tris containing
`0.05 molT 1 Na2HPO4 at pH 13.0
`and 0~8.0 moH- 1 urea
`
`0.1
`
`0
`
`2
`4
`6
`Urea concentration (mol ·I- 1)
`
`8
`
`10
`
`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
`introducing low concentration of urea in 0.05 molT 1 Tris buffer containing 0.05 molT 1
`Na2HPO4 at pH 13.0; an approximate plateau was approached when the urea concentration
`was enhanced to 2.0 molr 1
`•
`
`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,
`
`Fig. 3 Chromatogram of
`rhSCF refolded by IEC.
`Chromatographic conditions:
`gradient/linear gradient from 0%
`B to 100% Bin 30 min, with a
`delay of 10 min; flow rate is
`2.0 ml·min- 1
`; mobile phase A:
`0.05 molr 1 Tris (pH = 8.0),
`I mmoH- 1 EDTA, 1.0 mmol·l- 1
`GSH, 0.1 mmoH- 1 GSSG;
`mobile phase B: the mobile phase
`A containing I molr 1 NaCl; the
`asterisk denotes rhSCF
`
`e C:
`
`~ 0.10
`N
`8
`!a
`,E 0.05
`0
`</)
`.0
`<I'.
`
`•
`
`10
`
`t/min
`
`20
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`Appl Biochem Biotechnol (2008) 144:181- 189
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`Fig. 4 SOS- PAGE analysis
`of rhSCF. I rhSCF refolded by
`IEC with simultaneous
`purification; 2 extract of rhSCF
`inclusion bodies by high pH
`buffer containing 2.0 mol·l- 1 urea
`
`187
`
`1
`
`2
`
`IEC was used to refold rhSCF solubilized by high pH buffer containing 2.0 mo1T 1 urea;
`the chromatogram is shown in Fig. 3. The whole IEC refolding process could be
`accomplished in I h, including the equilibration and elution program. The obtained rhSCF
`has a specific bioactivity (SB) of7.8 x 105 IU·mg- 1
`, a mass recovery (MR) of43.0%, and a
`purity of 96.3% (Fig. 4). For comparison, the rhSCF solubilized by high pH buffer
`containing 2.0 molT 1 urea was also refolded by dilution and dialysis, and the above used
`three refolding methods were also applied to refold the rhSCF solubilized by 8.0 mol·l- 1
`urea. The results are shown in Table I. It can be seen from this table that all of the mass
`Table I Comparison of results for rhSCF solubilized and refolded by using a different method.
`rhSCF
`SB1EC
`sample
`(IU·mg- 1)
`
`rhSCfu,ea a
`rhSCfptt b
`
`(3.3 ±0.94) x 105
`18.8± 1.53
`(4.5± I.I) X 105
`(4.7 ±0.86) xt05
`25.4±2.16
`(5.4± 1.4) X 105
`a rhSCFu,ea presents the rhSCF solubilized by 8.0 mol·l- 1 urea.
`b rhSCFpH presents the rhSCF solubilized by 0.05 mol·J"1 Tris (pH I 3.0) containing 0.05 mol·l- 1 Na2HP04
`and 2.0 mol-1- 1 urea.
`
`16.8± 1.07
`26.2± 1.79
`
`(7.6± 1.8) x t05
`(7.8 ± 1.5) xJ05
`
`36.4±3.10
`43.0±2.93
`
`SBdilution
`(IU·mg- 1)
`
`MRdilution
`(%)
`
`SBdialysis
`(IU·mg- 1)
`
`MRdialysis
`(%)
`
`MRmc
`(%)
`
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`Appl Biochem Biotechnol (2008) 144:181- 189
`
`recovery for rhSCF solubilized by high pH buffer containing 2.0 moH- 1 urea are higher
`than that solubilized by 8.0 moH- 1 urea, no matter which refolding method was employed,
`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 I 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
`solubilized by 8 mo1T 1 urea solution, refolded and oxidized by dilution refolding with a
`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 Modem Separation Science in Shaanxi Province
`(no. 05JS6 l ).
`
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