Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aoba-yama 07, Aoba-ku, Sendai 980-8579, Japan
1 To whom correspondence should be addressed. e-mail: tsumoto{at}mail.cc.tohoku.ac.jp; kmiz{at}mail.cc.tohoku.ac.jp
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Abstract |
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Keywords: foldase/immobilization/immunoglobulin fold/oxidoreductase/refolding
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Introduction |
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Often, inclusion bodies consist mainly of the aggregated expressed proteins, with only minor contamination by lipids and nucleic acids. Thus, developing an efficient, convenient, economical method for refolding the aggregated protein into its native form would enable a large amount of functional protein to be recovered, sufficient for industrial yields.
We recently reported a highly efficient refolding process for immunoglobulin-folded proteins using stepwise dialysis (Tsumoto et al., 1998; Asano et al., 2002
a,b). The most critical factors for highly efficient recovery of proteins from a denatured and reduced state are prevention of aggregation by adding labilizing agents and promotion of proper disulfide bond formation at the appropriate stage of refolding (Figure 1) (Tsumoto et al., 1998
). When the concentration of guanidium hydrochloride (GdnHCl) was decreased by stepwise dialysis, L-arginine (L-Arg) and oxidized glutathione (GSSG) were used as a labilizing agent and an oxidant, respectively, to attain a highly efficient yield of refolded protein. Recently, we carefully analyzed the pathway of the refolding process using various spectroscopic techniques and found that the timing of adding the agents critically influences the formation of correct disulfide bonds (Umetsu et al., 2003
).
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Materials and methods |
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All enzymes for genetic engineering were obtained from Takara Shuzo (Kyoto, Japan), Toyobo (Osaka, Japan), Boehringer Mannheim (Mannheim, Germany) and New England Biolabs (Beverly, MA, USA). GdnHCl and isopropyl-ß-D-thiogalactopyranoside (IPTG) were obtained from Wako Fine Chemicals Inc. (Osaka, Japan). All other reagents were of biochemical research grade. The HyHEL-10 scFv [VLlinkerVH(His)6] was prepared as described previously (Umetsu et al., 2003).
Construction of GroEL apical domain, DsbA and DsbC expression plasmids
The gene encoding the GroEL apical domain (site 191345 of E.coli GroEL) was amplified by polymerase chain reaction (PCR), using 5'-NNNCATATGGAAGGTATGCAGTTCGA CCGTGGC-3' (NdeI site is underlined) and 5'-NNN GGATCCTTAACGGCCCTGGATTGCAGCTTC-3' (BamHI site is underlined) as primers and E.coli TG1 genome as template. The PCR product was digested with NdeI and BamHI, purified, and cloned into the NdeIBamHI site of pET-15b (Novagen, USA). Each dsb gene (plus ShineDalgarno sequence) was amplified by PCR using the E.coli TG1 genome as template and 5'-NNNCATATGAAAAAGATTTGGCTG GCG-3' (NdeI site is underlined) and 5'-NNNGGATCC TTATTTTTTCTCGGACAG-3' (BamHI site is underlined) as primers for dsbA and 5'-NNNCATATGAAGAAAGGT TTTATGTTGTTTACTTTG-3' and 5'-NNNGGATCCTTA TTTACCGCTGGTCATTTTTTGG-3' as primers for dsbC. The PCR product was digested with NdeI and BamHI, purified, and cloned into the NdeIBamHI site of pET-20b (Novagen).
Expression and preparation of the mini-chaperone, DsbA and DsbC
The expression and purification of the mini-chaperone were carried out as follows. E.coli strain BL21 (DE3) transformed with each expression vector was incubated at 28°C in LuriaBertani (LB) broth. When the optical density reached 0.8 at 600 nm, 0.1 mM IPTG was added to the culture to induce protein production and the cells were grown overnight. Cells were separated from 1 l of culture by centrifugation (2000 g, 35 min) and resuspended in 10 ml of 50 mM TrisHCl (pH 8.0) containing 150 mM NaCl. Cells were broken down by ultrasonication at 150 W for 15 min and centrifuged at 4500 g for 20 min. The soluble (formerly intracellular) fraction was passed over a Talon metal affinity resin column (Clontech, Palo Alto, CA, USA). Minor impurities were removed by gel filtration on a Sephacryl S-200 column pre-equilibrated and eluted with 50 mM TrisHCl (pH 8.0) containing 150 mM NaCl.
DsbA and DsbC were expressed as described above. Cell pellets were resuspended in 10 ml of 20 mM TrisHCl (pH 7.5), 0.5 M sucrose and 0.1 mM EDTA and incubated for 5 min at room temperature. Then, 40 ml of water was added to give an osmotic shock and the cells were left on ice for 30 min. The cells were collected by centrifugation at 7000 g for 60 min at 4°C. The supernatant was salted out with 80% saturated ammonium sulfate and the precipitate was collected by centrifugation at 7000 g for 30 min. The protein precipitate was dissolved in 50 mM TrisHCl (pH 8.0) containing 5 mM NaCl and was dialyzed against the same buffer for 2 days. The precipitate that formed during dialysis was removed by centrifugation at 10 000 g for 15 min. The supernatant was loaded onto a DEAESepharose FF (Amersham Biosciences, Tokyo, Japan) column and the eluate was subjected to gel filtration on a Sephacryl S-200 column pre-equilibrated and eluted with 50 mM TrisHCl (pH 8.0) containing 150 mM NaCl.
Immobilization of foldases
Wet N-hydroxysuccinimide-activated Sepharose 4FF (3 ml) (Amersham Biosciences) was washed with 50 ml of 1 mM HCl on an ice bath and then suspended in 50 mM NaHCO3 (pH 8.0) containing 500 mM NaCl (coupling buffer). Foldases, pre-equilibrated with the coupling buffer, were added to the regin suspension (10 mg of protein per 1 ml of regin) and mixed gently overnight at 4°C. The resin was washed with the coupling buffer and any remaining active groups were blocked with ethanolamine; then the resin was washed according to the manufacturers recommendations. The SH groups on the foldases were regenerated by incubation with 5 mM ß-mercaptoethanol (ß-ME) at 4°C for 1 h. The resin was washed with the buffer and stored at 4°C. More than 95% of the foldases in the solution became immobilized and the resins were stable for more than 1 year at 4°C.
Measurement of chaperone-like activity
The chaperone-like activity (i.e. prevention of aggregation) of the mini-chaperone, DsbA and DsbC was determined by using denatured scFv as the substrate protein: scFv (15 µM) was chemically denatured in 6 M GdnHCl, 1 mM ß-ME for more than 4 h at room temperature in the dark and the concentration of GdnHCl was brought to 2 M as described below. Enzyme activity was determined by the method of Umetsu et al. (2003).
Disulfide isomerase activity of soluble and immobilized Dsbs
The disulfide isomerase activity of soluble and immobilized DsbA and DsbC was assayed according to Lyles and Gilbert (1991), using scrambled bovine RNaseA as a substrate.
Ellman assay
DsbA, DsbC and scFv solutions were diluted to 1.5 µM protein concentration with 50 mM TrisHCl buffer and then 50 µl of 10 mM 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) solution (50 mM potassium phosphate buffer, pH 7.0, 1 mM EDTA) was added to 1.25 ml of the diluted protein solution. The number of free thiol groups was calculated from the monitoring of the absorbance at 412 nm after adding DTNB (Sadlak and Lindsay, 1968).
Refolding of antibody fragments expressed as inclusion bodies
Dilution method. Purified scFv fragments were denatured in 6 M GdnHCl, 1 mM ß-ME for more than 4 h at room temperature in the dark (scFv concentration, 15 µM) and then the concentration of denaturant was adjusted to 2 M by dialysis. The scFv solution was diluted to the final scFv and GdnHCl concentrations of 3 µM and 0.5 M, respectively, by a refolding solution (Wunderlich et al, 1994).
Stepwise dialysis method. The scFv fragments denatured in 6 M GdnHCl were reduced by adding 357 µM ß-ME (scFv concentration, 7.5 µM). The procedures for refolding insoluble antibody fragments were performed according to the method of Tsumoto et al. (1998) (stepwise dialysis method) except for adding the foldases instead of L-Arg and GSSG. According to the previous procedure (Tsumoto et al., 1998
), the concentration of GdnHCl in the dialysis buffer was phased down (3, 2, 1, 0.5 and 0 M). An oxidant (GSSG, DsbA or DsbC) and a reagent for preventing aggregate formation (0.4 M L-Arg or 375 µM mini-chaperone) were added when the dialysis buffer contained 1 or 0.5 M GdnHCl.
Functional characterization of refolded antibody fragments
After the refolding procedure described above, the supernatants containing refolded proteins were concentrated to 10 µM with a Centriplus YM-10 ultrafiltration membrane (Millipore, Bedford, MA, USA). The concentrated scFv was purified on a Superdex 75 pg (16 x 400 mm) column equilibrated with 50 mM TrisHCl (pH 7.5) containing 200 mM NaCl at 4°C, and dialyzed overnight against 50 mM phosphate buffer (pH 7.2) containing 200 mM NaCl. scFv binding was assayed essentially according to Ueda et al. (1993)
. Various concentrations of Fv or scFv were mixed with 1.5 µM hen lysozyme in 30 µl of phosphate-buffered saline and incubated at 25°C for 1 h. Each mixture was then added to 970 µl of 50 mM NaH2PO4 buffer (pH 6.20, adjusted with NaOH) containing 340 µg of Micrococcus luteus cells. The initial rate of decrease in absorbance at 540 nm was monitored at 25°C.
Definitions of refolding efficiency and inhibitory activity
Refolding efficiency has been defined as the amount of the soluble proteins after refolding relative to the total proteins subjected to the refolding procedure, which has been calculated from the protein concentration quantified by the BCA protein assay (Pierce). The inhibitory activity of the refolded protein toward the target protein, lysozyme, is relative to that of the secretory expressed (i.e. native) Fv fragment, which has been evaluated as described above. For instance, in the situation where the refolding efficiency and inhibitory activity are 50 and 100%, respectively, half of the proteins could be solubilized in the refolding buffer and the refolded soluble protein has almost identical inhibitory activity to the native Fv fragment.
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Results |
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Mini-chaperone, DsbA and DsbC were expressed and obtained as soluble proteins in final yields of 90,
26 and
80 mg/l of culture, respectively. Gel filtration of the mini-chaperone showed that it was monomeric; it was fully functional in refolding a typical substrate, denatured carbonic anhydrase (data not shown).
Free thiol groups in expressed and purified DsbA and DsbC constituted only 5% of all Cys groups, indicating that almost all the Dsbs were in the oxidized form. These disulfides were susceptible to excess reducing agent, e.g. dithiothreitol or ß-ME. The disulfide isomerase activities of DsbA and DsbC were 6 and 8% relative to bovine protein disulfide isomerase, respectively, in the oxidized form, and 5 and 30%, respectively, in the reduced form.
For immobilized foldases, careful treatment of free thiol groups under an Ar atmosphere led to functional immobilization of DsbA and DsbC (data not shown) (Altamirano et al., 1999). Free thiol groups in expressed and purified, immobilized DsbA and DsbC also constituted 5% of all Cys groups, indicating that almost all of the immobilized Dsbs were in the oxidized form.
Prevention of scFv aggregation by soluble mini-chaperone, DsbA and DsbC in the dilution method
Complete prevention of aggregation was achieved by dilution with the refolding buffer containing a 100-molar excess of the mini-chaperone to the substrate scFv (3 µM, Figure 2a) and also by a 10-molar excess of DsbC (Figure 2c). Incomplete prevention was observed at a 10-molar excess of DsbA (Figure 2b), whose activity was somewhat lower than that of DsbC (Figure 2c). L-Arg prevented aggregation at a concentration of 0.4 M (Figure 2d); GSSG and bovine serum albumin (BSA) did not prevent aggregation (data not shown). These results indicate that mini-chaperone, DsbA and DsbC have chaperone-like activity for scFv in a dose-dependent manner and that these foldases can recognize partially folded scFv as a substrate.
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We next examined the effects of foldases on scFv aggregation during refolding in stepwise dialysis. Adding mini-chaperone to the refolding solution together with GSSG prevented aggregation at a concentration of 750 µM (i.e. 100-molar excess) (Figure 3a). A 10-molar excess of DsbA and DsbC decreased aggregation (Figure 3b), indicating that DsbA and DsbC functioned even at a GdnHCl concentration of 1 M. However, the scFv aggregation could not be completely prevented even by adding L-Arg (Figure 3c). In contrast, the result of adding a 100-molar excess of the mini-chaperone to the refolding solution with DsbA completely prevented scFv aggregation, and furthermore, it should be noted for DsbC that the prevention of aggregation by the combination of the mini-chaperone (50-molar excess) with DsbC (10-molar excess) was more than just an additive effect caused by the coexistence of the mini-chaperone with DsbC. This suggests that the mini-chaperone in combination with oxidoreductases led to better refolding efficiency than with glutathione.
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Figure 4 shows the refolding efficiency and the functional recovery of scFv by application of immobilized foldases (mini-chaperone, DsbA and DsbC) to the stepwise dialysis method. The refolding efficiency is the ratio of the soluble fraction at the final stage in stepwise dialysis to the total amount of scFv used for refolding, and the functional recovery is estimated from the activity of the soluble scFv relative to that of native Fv. Addition of the mini-chaperone immobilized on agarose resin to the refolding solution along with GSSG only enhanced the refolding efficiency of scFv to a small degree in comparison with the case using only GSSG at each concentration of resin (samples ad in Figure 4). Furthermore, the refolding efficiency of scFv using the immobilized mini-chaperone was lower than that using the non-immobilized (i.e. soluble) mini-chaperone (Figure 3).
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Discussion |
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The soluble mini-chaperone prevented aggregation of scFv; however, a 100-molar excess of the mini-chaperone was required, and oxidants were required for functional recovery of the scFv. Goto and Hamaguchi (Goto and Hamaguchi, 1981) reported that spontaneous refolding did not lead to correct disulfide bond formation in the refolding of immunoglobulin-folded proteins without a redox shuffling system of glutathione, and they found that free sulfide groups caused protein aggregation. The application of eukaryotic protein disulfide isomerase (PDI) to the in vitro folding system was also reported to increase the reactivation of antibody (Lilie et al., 1994
), and recently, Mayer et al. (Mayer et al., 2000
) found that PDI acts synergistically with immunoglobulin heavy chain binding protein in the in vitro folding. Their report clearly supports our findings that both chaperone activity and disulfide bond formation are required for refolding immunoglobulin-folded proteins. When L-Arg is present during refolding, the introduction of GSSG at the 1 and 0.5 M GdnHCl stages of dialysis resulted in the best refolding yield (Umetsu et al., 2003
) However, in the presence of the mini-chaperone, the addition of an oxidoreductase, especially DsbC, resulted in a more complete binding activity than addition of GSSG (Table I), suggesting that disulfide bond shuffling occurred. The fact that the L-ArgDsbA (DsbC) refolding solution did not suppress aggregation of scFv might support the additional effect of the mini-chaperone for the oxidoreductase. Immobilization of GroEL is known to substantially decrease its chaperone activity (Taguchi et al., 1994
). In this study, we immobilized only the apical domain of GroEL, but the chaperone function was slightly decreased (samples bd in Figure 4).
DsbA prevented aggregation of scFv at the 1 M GdnHCl stage during the refolding by stepwise dialysis, while DsbC could not prevent aggregation at the same stage (Figure 3b). The chaperone-like activity of DsbA is evident from the finding that D-glyceraldehyde-3-phosphate dehydrogenase (GADPH) with triosephosphate isomerase (TIM) barrel fold can be refolded in the presence of DsbA despite it not having any disulfide bonds (Zheng et al., 1997). With scFv, both the correct disulfide bond formation and the correct immunoglobulin-fold formation are required. The results reported here indicate that DsbA recognizes the denatured or partially folded scFv as a substrate, although this is not enough to achieve a high yield of correctly refolded scFv.
DsbC prevented aggregation of scFv almost completely at a 10-molar excess of DsbC to scFv (Figure 2c), as suggested for GADPH (Chen et al., 1999). DsbC acts as a disulfide bond isomerase for scFv, indicating its usefulness for refolding immunoglobulin-folded proteins. Thus, our results indicate that, in solution, DsbC is more useful than DsbA for refolding scFv, probably because the isomerase activity of soluble DsbA is lower than that of DsbC (Zheng et al., 1997
; Chen et al., 1999
). However, immobilization of these oxidoreductases onto an agarose-based resin led to almost identical efficiencies in refolding scFv, perhaps due to an increase in the local concentration of DsbA.
Immobilization of oxidoreductases appeared to increase their stability dramatically: the activities of the immobilized isomerases were stable for more than a year, whereas these enzymes in solution do not retain activity for more than several months.
The inhibitory activity of scFv recovered from E.coli inclusion bodies with immobilized DsbC was almost the same as native Fv (sample n in Figure 4), although the refolding efficiency was 60%. In contrast, the refolding system with GSSG and L-Arg achieves 100% refolding efficiency, but the inhibitory activity of refolded scFv is 67% of the activity of native Fv, which is the same activity as secretory expressed scFv (sample f in Figure 4) (Tsumoto et al., 1994
; Umetsu et al., 2003
). Reconditioning activity has been proposed as a general property of refolding systems using immobilized foldases (Altamirano et al., 1997
; 1999
). Therefore, our results suggested that more functional immunoglobulin-folded proteins can be obtained with immobilized disulfide isomerases than utilization of GSSG and L-Arg, while the immobilized DsbC prevents less aggregation of scFv. Whether secretory expressed or refolded, soluble fraction of scFv prepared might include molecules with reduced binding activity, and thus, reconditioning activity of immobilized disulfide isomerases may be critical for preparation of functional proteins with intra- and inter-molecular disulfide bonds.
Our results demonstrate that immobilized oxidoreductases may have the following advantages for refolding immunoglobulin-folded proteins (scFv): (i) smaller volumes than dilution or dialysis methods are needed; (ii) there is less use of chemical reagents such as L-Arg and glutathione, which are hard to recycle; (iii) immobilized oxidoreductases are reusable without the need for complicated separation methods; and (iv) more homogenous scFv with the same activity as native Fv. Because many proteins that are needed in large amounts for therapeutics and diagnosis have disulfide bonds, immobilized oxidoreductases would enable batch processing. Stabilization of oxidoreductases (PDIs) via protein engineering or utilization of oxidoreductases (PDIs) from thermophiles [e.g. Pyrococcus furious (Guagliardi et al., 1995)] might improve immobilized oxidoreductases (PDIs). Finally, we would recommend the critical utilization of immobilized oxidoreductases as an effective additive for refolding several proteins with intra- and inter-molecular disulfide bonds.
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Acknowledgements |
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Received April 7, 2003; revised June 5, 2003; accepted June 6, 2003.