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Refolding of single-chain Fv by use of an antigen-coupled column
Biochemical Engineering Journal 44 (2009) 289–291
Contents lists available at ScienceDirect
Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Short communication
Refolding of single-chain Fv by use of an antigen-coupled column Kazutaka Ikeda a , Yoichi Kumada b , Tomohisa Katsuda a,∗ , Hideki Yamaji a , Shigeo Katoh a,c a
Department of Chemical Science and Engineering, Kobe University, Kobe 657-8501, Japan Department of Chemistry and Materials Technology, Kyoto Institute of Technology, 606-8585, Japan c Department of Chemical Engineering, Hanyang University, Ansan 426-791, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 29 August 2008 Received in revised form 15 December 2008 Accepted 28 December 2008 Keywords: Single-chain Fv Refolding Antigen-coupled column Aggregate
a b s t r a c t A single-chain variable fragment (scFv) antibody against bisphenol A was refolded using an antigen (bisphenol A)-coupled column. The refolding efficiency was compared with that used in dialysis. The refolding efficiency of the antigen-coupled column was about 50–60%, which was much higher than with dialysis, due to a decrease in the concentration of the refolding molecules and to the suppression of the aggregate formation. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Recombinant proteins over-expressed in Escherichia coli are often accumulated as insoluble particles called inclusion bodies. Since proteins in inclusion bodies usually are inactive, they must be solubilized by a denaturing agent and refolded to recover their native steric structures and, thereby, their biological activities. In a usual refolding process the concentration of the denaturing agent in a denatured protein solution is diluted either by batchwise dilution, dialysis or gel chromatography to start refolding. The refolding efficiency of these methods, however, is not always high enough for industrial production of recombinant proteins. In a previous study [1] the biospecific interaction between antigen and antibody was used for refolding in a packed column, for which an antibody against a target protein coupled with a gel support was used as a template ligand. With refolding in the column, a higher refolding efficiency was obtained in comparison with those obtained by the batch dilution method. Single-chain Fv (scFv) antibodies are artificial fusion proteins, in which Vh and Vl domains of an antibody are genetically linked by polypeptide linkers. These have wide applicability to immunoassay and drug discovery [2]. Although various scFvs are expressed with high productivity in recombinant E. coli cells, they are often recovered as inclusion bodies. Several refolding methods have been proposed for scFvs [3,4]. Utilization of antigen-coupled columns, however, may be useful due to their effective refolding, which gives
∗ Corresponding author. Tel.: +81 78 803 6207; fax: +81 78 803 6207. E-mail address: [email protected] (T. Katsuda). 1369-703X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2008.12.014
them general applicability because scFvs have their own antigens, or antigen analogs. In the present study a scFv that was produced by E. coli and denatured from a soluble fraction and inclusion body against an antigen bisphenol A, was refolded by use of a bisphenol A-coupled column. The refolding efficiency was then compared with that of the dialysis method. 2. Materials and methods 2.1. Materials The anti-bisphenol A scFv gene (Mouse), in which the Cterminus of the Vh domain and the N-terminus of the Vl domain were linked by a flexible linker (G4 S)3 , was used as a model antibody in this study [5]. HisTrap HP resin was purchased from GE Healthcare Bio-Sciences Corp. (Piscataway, NJ) and used for purification of the scFv. The bisphenol A-coupled Sepharose 6B was prepared following standard protocols from the supplier. Bisphenol A (100 mg) was dissolved in 10 ml of water by adjusting the pH to 11 using NaOH, which was followed by coupling to 1 g (dry base) of epoxyactivated Sepharosed 6B. All other reagents were of analytical grade unless otherwise specified. 2.2. Expression and purification of the scFv The gene of the scFv, which had a pel B signal sequence at the N-terminus and a (His)6 tag at the C-terminus, was ligated with the expression vector, pET 22b(+) (EMD Biosciences Inc., San Diego, CA) and transformed to BL21 (DE3) pLysS (EMD Biosciences Inc.).
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K. Ikeda et al. / Biochemical Engineering Journal 44 (2009) 289–291
Expression and purification of the scFvs were basically performed using the following procedures, as reported previously [5]. The transformed E. coli was inoculated into a 20 ml 2 × YT medium (+Amp, +Cam) in a 100-ml shake flask, and cultured overnight at 37 ◦ C and 130 rpm. Then, it was inoculated into 50 ml of 2 × YT medium (+Amp, +Cam) in a 500-ml shake flask at a final cell concentration of OD600 = 0.1. The flask was shaken at 200 rpm and 37 ◦ C for 4 h. Then, precultured cells were transferred to 1 l of the modified LCM 50 medium [6] in a 2-l jar fermentor (Mitsuwa KMJ5C, Japan) at a final cell concentration of OD600 = 0.2. Cells were cultured at 30 ◦ C with aeration at 4 VVM and agitation controlled from 400 to 1200 rpm in order to maintain the dissolved oxygen value at 4 ppm. The pH value was kept at 7.6 during cultivation by addition of 3 M hydrochloric acid or sodium hydroxide. When the cell concentration reached OD600 = 4.5–5.0, IPTG was added as an inducer at a final concentration of 1 mM. After E. coli cells re-started exponential growth, the feeding solution (513.4 g/l of glycerol, 135.5 g/l of ammonium sulfate and 1.0 g/l of ampicillin) was added to the culture medium at the flow rate (F(t)) determined by the equation reported by Korz et al. [6]—in order to maintain the growth of cells at a constant specific growth rate (in this work at 0.1 h−1 ). After centrifugation of the culture broth, the pellets were resuspended in lysis buffer containing 1% Triton X-100, 20 mM Tris–HCl (pH 8.0), 2 g/ml DNase, and 0.2 mg/ml lysozyme, and incubated under gentle stirring for 20 min at 37 ◦ C. To disrupt the cellular membrane, the suspension was sonicated for 20 min under cooling using a probe-type sonicator (TOMY, Tokyo). The suspension was ultra-centrifuged at 600,000 × g for 15 min. The supernatant and pellets were separated as a cellular soluble fraction and an insoluble fraction, respectively. The scFv in the cellular soluble fraction was purified by affinity chromatography using HisTrap HP and bisphenol A-coupled Sepharose 6B packed columns. The scFv bound to the bisphenol A-coupled Sepharose 6B column was eluted by 10 mM NaOH, and then the eluate was immediately neutralized with 1 ml of 2 M phosphate buffer (pH 7.2) followed by denaturation, as described below. The pellets of the insoluble fraction were washed 3 times with the lysis buffer by centrifugation at 23,500 × g for 5 min to remove membrane debris, and were further washed twice with distilled water followed by lyophilization before finally being used for denaturation of the inclusion body. 2.3. Denaturation and refolding The scFv in the soluble fraction was denatured in a denaturation buffer (8 M urea, 10 mM 2-mercaptetanol, 50 mM Tris–HCl, 0.2 M NaCl, pH8.0) at 25 ◦ C overnight. The inclusion body of the scFv was dissolved in the same denaturation buffer, and the supernatant was used for refolding after centrifugation at 15,000 rpm for 15 min. In dialysis refolding, 3 ml of denatured solution of the scFv was dialyzed against 500 ml of 50 mM Tris–HCl buffer (pH 8.0) for 3 h and centrifuged. The supernatant was applied to the bisphenol A-coupled column (1.5 cm i.d. and 1.8 cm bed height) after measurement of the protein concentration. The absorptive capacity of the active scFv decreased with an increase in the concentration of urea in the buffer solutions, but remained above 4 mg. The coupled fraction was eluted with 0.1 M NaOH, and the volume and protein concentration of the flow-through and eluted fractions were measured. In the antigen-coupled column refolding, the column was equilibrated with the 50 mM Tris–HCl buffer (pH 8.0), and the denatured scFv solution was diluted twice and supplied to a small mixing chamber at a flow rate of 0.2 ml/min with a micro-feeder pump (Furue Science Co., JP-V-W), then was mixed with the refolding buffer at a ratio of 1:1. Immediately after mixing, as shown in Fig. 1,
Fig. 1. Scheme of refolding using an antigen-coupled column.
the renaturation mixture was applied to the top of the bisphenol A-coupled column. After application, the outlet of the column was connected to the inlet tube, and the renaturation mixture was circulated with a micro-tube pump for 3 h. The recirculated liquid volume and the average urea concentration were about 9.5 ml and 0.8 M, respectively. After washing, adsorbed protein was eluted with 0.1 M NaOH. The flow-through and eluted fraction were dialyzed against PBS, and the volume and protein concentration were measured. The protein concentration in the eluate was determined using DC-protein assay (Bio-Rad Laboratories, CA) with BSA as a standard protein. The refolding efficiency is defined by the following equation: Refolding efficiency (%) =
amount of protein eluted × 100 amount of protein used for refolding
3. Results and discussion Fig. 2 shows the SDS-PAGE of denatured protein solutions before refolding (lanes 2 and 5) and the flow-through fractions (lanes 3 and 6) and eluted fractions (lanes 4 and 7) from the bisphenol A-coupled column after refolding with the column for soluble fraction and inclusion body, respectively. In the case of refolding from the soluble fraction, only the scFv band was observed at the position expected from the amino acid sequence of the scFv. On the other hand, the molecular weight was higher than expected in a denatured inclusion body solution. This may have been caused by incorrect processing of the pel B signal sequence, because over-expressed scFv created aggregates in the cytoplasm before processing. Denatured scFv from both soluble and inclusion bodies was refolded by the dialysis method and the bisphenol A-coupled column.
K. Ikeda et al. / Biochemical Engineering Journal 44 (2009) 289–291
Fig. 2. SDS-PAGE of samples in refolding using an antigen-coupled column (silver stained).
291
In the eluted fractions (lanes 4 and 7) only a single band corresponding to scFv was observed. This result means that non-specific adsorption of proteins on the column is slight, and that the active scFv was only adsorbed in the antigen column. On the other hand, very little protein was observed in flow-though fractions. It seemed that contaminating proteins existed in the denatured protein solutions and formed aggregates that were trapped by the packed gel layer by some sort of depth filtration. Fig. 3 compares the refolding efficiency of the dialysis method with that of the bisphenol A-coupled column. The refolding efficiency of the former method rapidly decreased when the concentration of the scFv in the refolding solutions was below 20%, irrespective of the soluble fraction and the inclusion body. On the other hand, the refolding efficiency of the antigen-coupled column was about 50–60%, which was much higher than the dialysis method. It has been reported that the gel matrix itself may improve refolding efficiency, but the effect was slight in the case of template refolding in an antigen–antibody interaction, as shown in previous work [1]. The addition of oxido-reducing agents such as GSH + GSSG had little effect on refolding efficiency both in dialysis and in antigen-column refolding. The coupling of refolded scFv with its antigen can be useful for improving refolding efficiency through the decreased concentration of refolding molecules in a refolding mixture and the suppression of the aggregate formation. This method should have wide application for the effective refolding of scFvs. References
Fig. 3. Refolding efficiency comparing dialysis with antigen-coupled column.
[1] S. Katoh, Y. Kumada, N. Maeshima, Template refolding by use of antibody-coupled affinity column, Chem. Eng. Technol. 28 (2005) 1394–1397. [2] A. Carrier, F.B. Ducancel, N. Settiawan, L. Cattolico, B. Maillere, M. Leonetti, P. Drevet, A. Menez, J.-C. Boulain, Recombinant antibody-alkaline phosphatase conjugates for diagnosis of human IgGs: application to anti-HBsAg detection, J. Immunol. Meth. 181 (1995) 177–186. [3] K. Tsumoto, K. Shinoki, H. Kondo, M. Uchikawa, T. Juji, I. Kumagai, Highly efficient recovery of functional single-chain Fv fragments from inclusion bodies overexpressed in Escherichia coli by controlled introduction of oxidizing reagent—application to a human single-chain Fv fragment, J. Immunol. Meth. 219 (1998) 119–129. [4] L.-H. Chen, Q. Huang, L. Wan, L.-Y. Zeng, S.-F. Li, Y.-P. Li, X.-F. Lu, Expression, purification, and in vitro refolding of a humanized single-chain Fv antibody against human CTLA4 (CD152), Protein Expr. Purif. 46 (2006) 495–502. [5] Y. Kumada, T. Kawasaki, Y. Kikuchi, S. Katoh, Polypeptide linkers suitable for the efficient production of dimeric scFv in Escherichia coli, Biochem. Eng. J. 35 (2007) 158–165. [6] D.J. Korz, U. Rinas, K. Hellmuth, E.A. Sanders, W.D. Deckwer, Simple fed-batch technique for high cell density cultivation of Escherichia coli, J. Biotechnol. 39 (1995) 59–65.
Contents lists available at ScienceDirect
Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Short communication
Refolding of single-chain Fv by use of an antigen-coupled column Kazutaka Ikeda a , Yoichi Kumada b , Tomohisa Katsuda a,∗ , Hideki Yamaji a , Shigeo Katoh a,c a
Department of Chemical Science and Engineering, Kobe University, Kobe 657-8501, Japan Department of Chemistry and Materials Technology, Kyoto Institute of Technology, 606-8585, Japan c Department of Chemical Engineering, Hanyang University, Ansan 426-791, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 29 August 2008 Received in revised form 15 December 2008 Accepted 28 December 2008 Keywords: Single-chain Fv Refolding Antigen-coupled column Aggregate
a b s t r a c t A single-chain variable fragment (scFv) antibody against bisphenol A was refolded using an antigen (bisphenol A)-coupled column. The refolding efficiency was compared with that used in dialysis. The refolding efficiency of the antigen-coupled column was about 50–60%, which was much higher than with dialysis, due to a decrease in the concentration of the refolding molecules and to the suppression of the aggregate formation. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Recombinant proteins over-expressed in Escherichia coli are often accumulated as insoluble particles called inclusion bodies. Since proteins in inclusion bodies usually are inactive, they must be solubilized by a denaturing agent and refolded to recover their native steric structures and, thereby, their biological activities. In a usual refolding process the concentration of the denaturing agent in a denatured protein solution is diluted either by batchwise dilution, dialysis or gel chromatography to start refolding. The refolding efficiency of these methods, however, is not always high enough for industrial production of recombinant proteins. In a previous study [1] the biospecific interaction between antigen and antibody was used for refolding in a packed column, for which an antibody against a target protein coupled with a gel support was used as a template ligand. With refolding in the column, a higher refolding efficiency was obtained in comparison with those obtained by the batch dilution method. Single-chain Fv (scFv) antibodies are artificial fusion proteins, in which Vh and Vl domains of an antibody are genetically linked by polypeptide linkers. These have wide applicability to immunoassay and drug discovery [2]. Although various scFvs are expressed with high productivity in recombinant E. coli cells, they are often recovered as inclusion bodies. Several refolding methods have been proposed for scFvs [3,4]. Utilization of antigen-coupled columns, however, may be useful due to their effective refolding, which gives
∗ Corresponding author. Tel.: +81 78 803 6207; fax: +81 78 803 6207. E-mail address: [email protected] (T. Katsuda). 1369-703X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2008.12.014
them general applicability because scFvs have their own antigens, or antigen analogs. In the present study a scFv that was produced by E. coli and denatured from a soluble fraction and inclusion body against an antigen bisphenol A, was refolded by use of a bisphenol A-coupled column. The refolding efficiency was then compared with that of the dialysis method. 2. Materials and methods 2.1. Materials The anti-bisphenol A scFv gene (Mouse), in which the Cterminus of the Vh domain and the N-terminus of the Vl domain were linked by a flexible linker (G4 S)3 , was used as a model antibody in this study [5]. HisTrap HP resin was purchased from GE Healthcare Bio-Sciences Corp. (Piscataway, NJ) and used for purification of the scFv. The bisphenol A-coupled Sepharose 6B was prepared following standard protocols from the supplier. Bisphenol A (100 mg) was dissolved in 10 ml of water by adjusting the pH to 11 using NaOH, which was followed by coupling to 1 g (dry base) of epoxyactivated Sepharosed 6B. All other reagents were of analytical grade unless otherwise specified. 2.2. Expression and purification of the scFv The gene of the scFv, which had a pel B signal sequence at the N-terminus and a (His)6 tag at the C-terminus, was ligated with the expression vector, pET 22b(+) (EMD Biosciences Inc., San Diego, CA) and transformed to BL21 (DE3) pLysS (EMD Biosciences Inc.).
290
K. Ikeda et al. / Biochemical Engineering Journal 44 (2009) 289–291
Expression and purification of the scFvs were basically performed using the following procedures, as reported previously [5]. The transformed E. coli was inoculated into a 20 ml 2 × YT medium (+Amp, +Cam) in a 100-ml shake flask, and cultured overnight at 37 ◦ C and 130 rpm. Then, it was inoculated into 50 ml of 2 × YT medium (+Amp, +Cam) in a 500-ml shake flask at a final cell concentration of OD600 = 0.1. The flask was shaken at 200 rpm and 37 ◦ C for 4 h. Then, precultured cells were transferred to 1 l of the modified LCM 50 medium [6] in a 2-l jar fermentor (Mitsuwa KMJ5C, Japan) at a final cell concentration of OD600 = 0.2. Cells were cultured at 30 ◦ C with aeration at 4 VVM and agitation controlled from 400 to 1200 rpm in order to maintain the dissolved oxygen value at 4 ppm. The pH value was kept at 7.6 during cultivation by addition of 3 M hydrochloric acid or sodium hydroxide. When the cell concentration reached OD600 = 4.5–5.0, IPTG was added as an inducer at a final concentration of 1 mM. After E. coli cells re-started exponential growth, the feeding solution (513.4 g/l of glycerol, 135.5 g/l of ammonium sulfate and 1.0 g/l of ampicillin) was added to the culture medium at the flow rate (F(t)) determined by the equation reported by Korz et al. [6]—in order to maintain the growth of cells at a constant specific growth rate (in this work at 0.1 h−1 ). After centrifugation of the culture broth, the pellets were resuspended in lysis buffer containing 1% Triton X-100, 20 mM Tris–HCl (pH 8.0), 2 g/ml DNase, and 0.2 mg/ml lysozyme, and incubated under gentle stirring for 20 min at 37 ◦ C. To disrupt the cellular membrane, the suspension was sonicated for 20 min under cooling using a probe-type sonicator (TOMY, Tokyo). The suspension was ultra-centrifuged at 600,000 × g for 15 min. The supernatant and pellets were separated as a cellular soluble fraction and an insoluble fraction, respectively. The scFv in the cellular soluble fraction was purified by affinity chromatography using HisTrap HP and bisphenol A-coupled Sepharose 6B packed columns. The scFv bound to the bisphenol A-coupled Sepharose 6B column was eluted by 10 mM NaOH, and then the eluate was immediately neutralized with 1 ml of 2 M phosphate buffer (pH 7.2) followed by denaturation, as described below. The pellets of the insoluble fraction were washed 3 times with the lysis buffer by centrifugation at 23,500 × g for 5 min to remove membrane debris, and were further washed twice with distilled water followed by lyophilization before finally being used for denaturation of the inclusion body. 2.3. Denaturation and refolding The scFv in the soluble fraction was denatured in a denaturation buffer (8 M urea, 10 mM 2-mercaptetanol, 50 mM Tris–HCl, 0.2 M NaCl, pH8.0) at 25 ◦ C overnight. The inclusion body of the scFv was dissolved in the same denaturation buffer, and the supernatant was used for refolding after centrifugation at 15,000 rpm for 15 min. In dialysis refolding, 3 ml of denatured solution of the scFv was dialyzed against 500 ml of 50 mM Tris–HCl buffer (pH 8.0) for 3 h and centrifuged. The supernatant was applied to the bisphenol A-coupled column (1.5 cm i.d. and 1.8 cm bed height) after measurement of the protein concentration. The absorptive capacity of the active scFv decreased with an increase in the concentration of urea in the buffer solutions, but remained above 4 mg. The coupled fraction was eluted with 0.1 M NaOH, and the volume and protein concentration of the flow-through and eluted fractions were measured. In the antigen-coupled column refolding, the column was equilibrated with the 50 mM Tris–HCl buffer (pH 8.0), and the denatured scFv solution was diluted twice and supplied to a small mixing chamber at a flow rate of 0.2 ml/min with a micro-feeder pump (Furue Science Co., JP-V-W), then was mixed with the refolding buffer at a ratio of 1:1. Immediately after mixing, as shown in Fig. 1,
Fig. 1. Scheme of refolding using an antigen-coupled column.
the renaturation mixture was applied to the top of the bisphenol A-coupled column. After application, the outlet of the column was connected to the inlet tube, and the renaturation mixture was circulated with a micro-tube pump for 3 h. The recirculated liquid volume and the average urea concentration were about 9.5 ml and 0.8 M, respectively. After washing, adsorbed protein was eluted with 0.1 M NaOH. The flow-through and eluted fraction were dialyzed against PBS, and the volume and protein concentration were measured. The protein concentration in the eluate was determined using DC-protein assay (Bio-Rad Laboratories, CA) with BSA as a standard protein. The refolding efficiency is defined by the following equation: Refolding efficiency (%) =
amount of protein eluted × 100 amount of protein used for refolding
3. Results and discussion Fig. 2 shows the SDS-PAGE of denatured protein solutions before refolding (lanes 2 and 5) and the flow-through fractions (lanes 3 and 6) and eluted fractions (lanes 4 and 7) from the bisphenol A-coupled column after refolding with the column for soluble fraction and inclusion body, respectively. In the case of refolding from the soluble fraction, only the scFv band was observed at the position expected from the amino acid sequence of the scFv. On the other hand, the molecular weight was higher than expected in a denatured inclusion body solution. This may have been caused by incorrect processing of the pel B signal sequence, because over-expressed scFv created aggregates in the cytoplasm before processing. Denatured scFv from both soluble and inclusion bodies was refolded by the dialysis method and the bisphenol A-coupled column.
K. Ikeda et al. / Biochemical Engineering Journal 44 (2009) 289–291
Fig. 2. SDS-PAGE of samples in refolding using an antigen-coupled column (silver stained).
291
In the eluted fractions (lanes 4 and 7) only a single band corresponding to scFv was observed. This result means that non-specific adsorption of proteins on the column is slight, and that the active scFv was only adsorbed in the antigen column. On the other hand, very little protein was observed in flow-though fractions. It seemed that contaminating proteins existed in the denatured protein solutions and formed aggregates that were trapped by the packed gel layer by some sort of depth filtration. Fig. 3 compares the refolding efficiency of the dialysis method with that of the bisphenol A-coupled column. The refolding efficiency of the former method rapidly decreased when the concentration of the scFv in the refolding solutions was below 20%, irrespective of the soluble fraction and the inclusion body. On the other hand, the refolding efficiency of the antigen-coupled column was about 50–60%, which was much higher than the dialysis method. It has been reported that the gel matrix itself may improve refolding efficiency, but the effect was slight in the case of template refolding in an antigen–antibody interaction, as shown in previous work [1]. The addition of oxido-reducing agents such as GSH + GSSG had little effect on refolding efficiency both in dialysis and in antigen-column refolding. The coupling of refolded scFv with its antigen can be useful for improving refolding efficiency through the decreased concentration of refolding molecules in a refolding mixture and the suppression of the aggregate formation. This method should have wide application for the effective refolding of scFvs. References
Fig. 3. Refolding efficiency comparing dialysis with antigen-coupled column.
[1] S. Katoh, Y. Kumada, N. Maeshima, Template refolding by use of antibody-coupled affinity column, Chem. Eng. Technol. 28 (2005) 1394–1397. [2] A. Carrier, F.B. Ducancel, N. Settiawan, L. Cattolico, B. Maillere, M. Leonetti, P. Drevet, A. Menez, J.-C. Boulain, Recombinant antibody-alkaline phosphatase conjugates for diagnosis of human IgGs: application to anti-HBsAg detection, J. Immunol. Meth. 181 (1995) 177–186. [3] K. Tsumoto, K. Shinoki, H. Kondo, M. Uchikawa, T. Juji, I. Kumagai, Highly efficient recovery of functional single-chain Fv fragments from inclusion bodies overexpressed in Escherichia coli by controlled introduction of oxidizing reagent—application to a human single-chain Fv fragment, J. Immunol. Meth. 219 (1998) 119–129. [4] L.-H. Chen, Q. Huang, L. Wan, L.-Y. Zeng, S.-F. Li, Y.-P. Li, X.-F. Lu, Expression, purification, and in vitro refolding of a humanized single-chain Fv antibody against human CTLA4 (CD152), Protein Expr. Purif. 46 (2006) 495–502. [5] Y. Kumada, T. Kawasaki, Y. Kikuchi, S. Katoh, Polypeptide linkers suitable for the efficient production of dimeric scFv in Escherichia coli, Biochem. Eng. J. 35 (2007) 158–165. [6] D.J. Korz, U. Rinas, K. Hellmuth, E.A. Sanders, W.D. Deckwer, Simple fed-batch technique for high cell density cultivation of Escherichia coli, J. Biotechnol. 39 (1995) 59–65.