How Additives Influence the Refolding of Immunoglobulin-folded Proteins in a Stepwise Dialysis System

SPECTROSCOPIC EVIDENCE FOR HIGHLY EFFICIENT REFOLDING OF A SINGLE-CHAIN FV FRAGMENT*

Mitsuo UmetsuDagger §, Kouhei TsumotoDagger , Masaki HaraDagger , Kumar AshishDagger , Shuichiro GodaDagger , Tadafumi Adschiri§, and Izumi KumagaiDagger ||

From the Dagger  Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aobayama 07, Aoba-ku, Sendai 980-8579 and § Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, 2-1-1, Katahira, Aoba-ku, Sendai 980-8577, Japan

Received for publication, December 2, 2002, and in revised form, January 7, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The gradual removal of the denaturing reagent guanidine HCl (GdnHCl) using stepwise dialysis with the introduction of an oxidizing reagent and L-arginine resulted in the highly efficient refolding of various denatured single-chain Fv fragments (scFvs) from inclusion bodies expressed in Escherichia coli. In this study, the influence of the additives on the intermediates in scFv refolding was carefully analyzed on the basis of the stepwise dialysis, and it was revealed that the additive effect critically changes the pathway of scFv refolding. Circular dichroism and tryptophan fluorescence emission spectroscopies demonstrated that distinct secondary and tertiary structures were formed upon dialysis from 2 M GdnHCl to 1 M GdnHCl, and 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid dipotassium salt binding analysis indicated that the addition of L-arginine to the stepwise dialysis system effectively stabilized the exposed hydrophobic area on the scFv. Quantification of the free thiol groups in the scFv by means of Ellman's assay revealed that there was a particular stage in which most of the free thiol groups were oxidized and that adding an oxidizing reagent (the oxidized form of glutathione, GSSG) at that stage was important for complete refolding of the scFv. The particular stage depended on the nature of the refolding solution, especially on whether L-arginine was present. Spontaneous folding at the 1 M GdnHCl stage resulted in a structure in which a free thiol group accessed to the proper one for correct disulfide linkage; however, the addition of L-arginine resulted in the formation of a partially folded intermediate without disulfide linkages. Mass spectrometry experiments on alkylated scFv were carried out at each stage to determine the effects of L-arginine. The spectroscopic studies revealed two different pathways for scFv refolding in the stepwise dialysis system, pathways that depended on whether L-arginine was present. Controlled coupling of the effects of GSSG and L-arginine led to the complete refolding of scFv in the stepwise dialysis.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The internal disulfide linkage in the immunoglobulin fold is of particular importance for most proteins in the immunoglobulin superfamily because this linkage has a critical influence on the stability of these proteins (1, 2). The linkage, which is highly conserved, connects two beta -sheets in a sandwich structure (3-6). In the case of antibody molecules, Goto and Hamaguchi (1, 7) were the first to analyze intrachain disulfide bond formation in the constant fragments of the immunoglobulin light chain (CL),1 demonstrating the contribution of the disulfide linkage to the stabilization and folding of the CL domain. For the variable fragment in antibody molecules (Fv), which is composed of heavy and light chain domains (VH and VL, respectively), the immunoglobulin fold is known to be partially and irreversibly denatured to an aggregate under reducing conditions (2); furthermore, the instability of the Fv that is missing its disulfide linkages has been proved by natural antibodies missing a disulfide linkage (8) and replacement of the essential cysteinyl residue by serine residue (9). Recently, the folding kinetics and aggregation behavior of the single-chain Fv fragment (scFv) after removal of the disulfide linkages have been analyzed by fluorescence spectroscopy and a combination of mass spectrometry and an H/D exchange technique (10, 11). In the folding of the disulfide-containing scFv in vitro, the presence of the disulfide linkage accelerates the independent folding of the VH and VL domains, and then the domains are associated with each other to form the correct quaternary structure. For the scFv with a domain containing no cysteinyl residues, the disulfide-free domain refolds more slowly than the disulfide-containing domain in the same scFv, and refolding requires an interface on the domain with a native disulfide linkage to form a functional structure (10). It has also been noted that when VH folds prior to interaction with the VL domain, the native structure results, whereas when the stable VL domain is formed before VH, aggregate formation is likely (10).

Antibody molecules are one of the key proteins for diagnostics in medicine and various researches in the field of proteomics (12, 13). Bacterial expression systems for recombinant proteins, especially systems using Escherichia coli, have been utilized to produce tailor-made antibody molecules such as scFv and Fab fragments (14, 15). However, the expression of antibodies in E. coli is usually limited to the periplasm because the reducing condition in the cytoplasm rules out the formation of disulfide linkages that is crucial for the immunoglobulin fold. Furthermore, the secretory expression of most scFv and Fab fragments involves insoluble aggregated forms called inclusion bodies in the periplasm (14, 16), and some antibody fragments cannot be expressed even as inclusion bodies (17). Consequently, a few other systems have been developed for the production of functional antibodies from E. coli, for example, direct production of functional scFv in the cytoplasm by the coexpression of disulfide bond chaperones (18), and refolding of recombinant antibody fragments from inclusion bodies in vitro (19).

The coexpression system is a recent development, but the refolding approach has been utilized for a decade to renature scFv and Fab fragments from their aggregated forms (2, 19-22). The general refolding system is based on dilution of denatured protein in a refolding buffer, and thiol-disulfide interchange reaction is applied to the system in order to increase the yield of refolding. However, the dilution method does not always result in a high refolding yield, and a more efficient method is needed.

Recently, we proposed (23) an improved refolding system for completely unfolded antibody molecules with reduced cysteinyl residues, a system based on the gradual removal of the denaturing reagent by means of stepwise dialysis. The refolding yield and the recovery of biological activity are greater than 95% in the case of various scFvs. Disulfide exchange and suppression of protein aggregation are controlled by the addition of the oxidized form of glutathione (GSSG) and L-arginine at appropriate stages (23). The high refolding yield, however, has not yet been evaluated in the view of physical and chemical studies, which are critically important for elucidating the stepwise folding process that leads to the completely functional scFv and for understanding how GSSG and L-arginine influence the folding pathway.

In this paper, we present spectroscopic studies on the refolding process of the scFv from a mouse anti-lysozyme monoclonal antibody, HyHEL-10, in our stepwise dialysis system. We measured CD and tryptophan fluorescence emission spectra for the scFv solution at each stage to observe the conformational changes in the secondary and tertiary structures. We also used 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid dipotassium salt (bis-ANS) analysis to probe hydrophobic area formation on scFv during the dialysis, and the analysis indicated a correlation between aggregation and the formation of an exposed hydrophobic area on scFv. Quantification of the free thiol groups in scFv, by means of Ellman's assay and mass spectrometry experiments, indicated that the pathway of disulfide bond formation in the presence of L-arginine was essentially different from the pathway in the solution containing only GdnHCl. The secondary structure and the number of free thiol groups for an aggregated scFv in the stepwise dialysis system were estimated by Fourier transform infrared (FT-IR) spectroscopy and Ellman's assay, and we discuss the factors leading to the formation of insoluble aggregates at each stage. The folding pathway for scFv depended strongly on the additives (GSSG and L-arginine), and the addition of these reagents at a particular stage enabled scFv to be refolded in high yield with recovery of biological activity.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- The plasmid containing the HyHEL-10 scFv (VL-linker-VH-(His)6) cDNA was constructed by insertion of the NcoI-XbaI fragment of pUTN3 (24) into the NcoI-XbaI-digested plasmid pUT7 (25). Transformed E. coli BL21 (DE3) cells were incubated in 2× YT medium at 28 °C, and expression of the HyHEL-10 scFv under the control of the T7 promoter was induced by adding 1 mM isopropyl-beta -D-thiogalactopyranoside. The harvested cells were centrifuged and suspended in 50 mM Tris-HCl (pH 8.0) buffer with 200 mM NaCl. After sonication, the suspension was centrifuged at 5,800 × g for 30 min at 4 °C. The pellet was suspended several times in the 50 mM Tris-HCl (pH 8.0) buffer with 4% Triton X-100 and 200 mM NaCl to remove nonspecifically adsorbed proteins, and the solution was centrifuged again at 5,800 × g for 30 min at 4 °C (23). After the removal step was repeated, the pellet was washed in water to remove Triton X-100, and then it was solubilized in the 50 mM Tris-HCl (pH 8.0) buffer with M GdnHCl and 200 mM NaCl. The solubilized scFv was refined by means of 1) a metal-chelate chromatography column that interacted with the histidine tag in scFv and 2) a gel filtration chromatography column (Sephacryl S-200).

Refolding of HyHEL-10 scFv by Stepwise Dialysis (23)-- The concentration of the unfolded HyHEL-10 scFv was adjusted to 7.5 µM in the 50 mM Tris-HCl (pH 8.0) buffer with 6 M GdnHCl, 200 mM NaCl, and 1 mM EDTA, and the fragment was reduced by the addition of 2-mercaptoethanol (beta -ME) at a 50-fold molar excess relative to the protein. After beta -ME was removed by dialysis against the same Tris-HCl buffer without beta -ME, the unfolded scFv was refolded by gradual removal of GdnHCl by means of stepwise dialysis from 6 to 0 M through 3, 2, 1, and 0.5 M by one of the following four methods (Fig. 1): (a) the concentration of GdnHCl was decreased by stepwise dialysis without any additives; (b) GSSG (50-fold molar excess relative to protein) was added at the 1 and 0.5 M GdnHCl stages; (c) 400 mM L-arginine was added at the 1 and 0.5 M GdnHCl stages; and (d) GSSG (50-fold molar excess relative to protein) and L-arginine (400 mM) were added at the 1 and 0.5 M GdnHCl stages.


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 1.   The stepwise dialysis system.

Absorption, CD, Fluorescence, and FT-IR Spectroscopy Experiments-- Absorption spectra were measured on a U-3000 spectrophotometer (Hitachi Ltd., Japan) in a 1-cm cuvette. CD spectra were measured on a J-720w spectropolarimeter (Jasco Inc. Japan) as follows: path length, 1.0 mm; bandwidth, 1.0 nm; resolution, 0.1 nm; response time, 8 s; scan speed, 5 nm/min; and number of scans, 4. The spectra of the scFv solutions at each stage of the dialysis were directly measured, but the CD spectra of the solution containing aggregated particles were measured after the removal of the particles by centrifugation.

Fluorescence spectra were recorded for each stage with an RF-5300PC spectrofluorophotometer (Shimadzu Co. Japan) in a 1-cm quartz cuvette. Tryptophan fluorescence emission spectra were measured for the scFv solution at a concentration of 0.5 µM with an excitation wavelength of 280 nm, and bis-ANS fluorescence spectra were measured at an excitation wavelength of 395 nm 10 min after bis-ANS was mixed with scFv in a 1:10 ratio (bis-ANS, 0.38 µM; scFv, 3.8 µM). The scFv solutions containing aggregated particles were utilized after the removal of the particles by centrifugation.

Diffuse reflectance FT-IR spectra were obtained on an FTS-165 (Bio-Rad); the aggregated form of scFv was dried under vacuum and then mixed with KBr at about 1% (w/w).

Ellman's Assay (26)-- Ellman's assay was applied to the scFv solutions at each stage, and the scFv was denatured by the dialysis from the scFv solutions at each stage to 6 M GdnHCl solution (100 mM potassium phosphate HCl (pH 5), 1 mM EDTA). Each scFv solution was diluted to 1.5 µM with the Tris-HCl buffer keeping each GdnHCl concentration after aggregated particles had been removed by centrifugation, and then 50 µl of 10 mM DTNB solution (50 mM potassium phosphate buffer, pH 7.0, 1 mM EDTA) was added to 1.25 ml of the diluted scFv solution. The absorbance at 412 nm was monitored after the addition of DTNB, and the number of the free thiol groups in an scFv was calculated from the extinction coefficient of nitrothiophenol, which is generated by reaction of DTNB with a thiol group. The exact extinction coefficient of nitrothiophenol was estimated in each solution by reaction with the reduced form of GSH because the coefficient depended on the concentrations of GdnHCl and L-arginine.

For quantification of the free thiol groups in the aggregated scFv in the stepwise dialysis, the aggregated particles were collected by centrifugation at each stage so that the insoluble particles aggregated at the different stage did not become mixed, e.g. the dialysis from 1 M GdnHCl to 0.5 M was carried out after the removal of the aggregated particles formed at the 1 M stage. The aggregated scFv at each stage was dissolved in 6 M GdnHCl at pH 5 before the assay was carried out.

Alkylation of the scFv at Each Stage in the Stepwise Dialysis-- An iodoacetic acid solution (50 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA) was added to the scFv solution obtained from each stage (final concentrations: iodoacetic acid, 1 mM; scFv, 5 µM), and the pH was adjusted to 8 with an NaOH solution. After 30 min, the alkylated scFv solution was dialyzed to 2 M GdnHCl to remove the residual iodoacetic acid and to standardize the concentration of GdnHCl. For alkylation of the scFv in the denatured state, the scFv solutions from each stage were dialyzed to 6 M GdnHCl (100 mM potassium phosphate-HCl, pH 5, 1 mM EDTA), and then the iodoacetic acid solution was added to the denatured scFv solutions.

Mass Spectrometry-- Mass spectra were measured on a REFLEX III matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometer (Bruker Analytische, GmbH, Germany) equipped with a nitrogen laser (337 nm). Sinapic acid was applied as a matrix and was dissolved to saturation in water/acetonitrile (2:1, v/v) containing 0.067% trifluoroacetic acid. Sample solutions from each stage were mixed with the sinapic acid-saturated solution in a 1:1 (v/v) ratio, and then 1 µl of the mixed solution was loaded onto the sample target. After co-crystallization on the target, the crystals were washed 2 times with 2 µl of water containing 0.1% trifluoroacetic acid to remove GdnHCl and other salts. Analysis was performed in positive and linear modes with an accelerating voltage of 27 kV, and 200 scans were averaged. The spectra obtained were calibrated externally using the [M + H+] ions from two protein standards: cytochrome c from horse heart (m/z 12360.08) and bovine trypsin (m/z 23311.53).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Aggregate Growth in the Stepwise Dialysis System-- We used the absorbance at 320 nm as a probe for suspension of scFv in the stepwise dialysis system (Fig. 2). No suspension was observed in solutions at GdnHCl concentrations higher than M. A rapid increase in the 320 nm absorbance was observed at the 1 M GdnHCl stage only for the simple GdnHCl solution, i.e. the solution without the GSSG and L-arginine additives (Fig. 2); this increase indicated aggregation of scFv in the solution. In contrast, no scFv aggregates formed at the 1 M GdnHCl stage in the solutions with GSSG or L-arginine (Fig. 2). These additives suppressed aggregation in the dialysis from 2 to 1 M. At the 0.5 M GdnHCl stage, aggregation was observed in all the solutions except for the optimal GdnHCl solution, which contained both GSSG and L-arginine (Fig. 2), although the additives promoted the folding of scFv to some extent. The aggregation of scFv at the 0.5 M GdnHCl stage of stepwise dialysis could be suppressed only by the addition of both GSSG and L-arginine.


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 2.   Change in the absorbance at 320 nm for the scFv solution at each stage in the stepwise dialysis. Simple GdnHCl solution (solid squares), GdnHCl-GSSG solution (open triangles), GdnHCl-L-arginine solution (solid triangles), and optimal GdnHCl solution (open squares).

Formation of Secondary and Tertiary Structures-- Fig. 3 shows the CD spectra of scFv in the simple GdnHCl solution at each stage of the stepwise dialysis. In the 6 M GdnHCl solution, the CD spectrum of scFv showed no characteristic CD signals derived from secondary structure in the region of 215-225 nm and instead showed a rather large trough with a tail extending beyond 220 nm, which implies random structure (Fig. 3a). Spectra with extended tails were also observed for the 3 and 2 M GdnHCl solutions, and no secondary structure was observed (Fig. 3, b and c). For the solutions of scFv in 1, 0.5, and 0 M GdnHCl, the CD spectra were measured after insoluble scFv aggregates were removed by centrifugation. Although the CD spectra still indicated random structure in the 1 and 0.5 M GdnHCl solutions, the random component got small around 210-220 nm, and a new trough appeared at 218 nm (Fig. 3, d and e). The 218-nm trough could be derived from a polypeptide with beta -strand structure. In the 0 M GdnHCl solution, in which scFv was folded, the 218-nm trough was also observed, and there was no CD signal for random structure (Fig. 3f). Therefore, the secondary structure of scFv was mainly formed upon dialysis from the 2 M GdnHCl solution, and the refolded scFv has a structure rich in beta -strands. The CD spectrum of scFv at the 1 M GdnHCl stage was also measured in the GdnHCl solution with GSSG (Fig. 4). Comparison of the spectra in the GdnHCl solution with those in GdnHCl-GSSG solution clearly showed that more beta -strand structure was formed in the presence of GSSG. Because L-arginine has strong CD intensity in the measured region, we could not measure the CD spectra of scFv in the GdnHCl solutions with L-arginine.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 3.   CD spectra of scFv in the simple GdnHCl solution. a, 6 M; b, 3 M; c, 2 M; d, 1 M; e, 0.5 M; f, 0 M. At the 0 M GdnHCl stage, the concentration of scFv was low, because of aggregation, and therefore the CD spectrum was measured after the solution was concentrated by ultrafiltration.


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 4.   CD spectra of scFv at the 1 M GdnHCl stage in the simple GdnHCl solution (solid line) and the GdnHCl-GSSG solution (dashed line).

The folding of scFv in the dialysis system was also explored by means of the change in the tryptophan fluorescence emission maximum (Fig. 5). The scFv has six tryptophan residues (four in VH and two in VL), and all the residues except for Trp-95 in VH are buried inside the native structure (27). The tryptophan molecules in scFv showed an intense emission with a maximum at 353 nm in the 6 M GdnHCl solution, and the emission was slightly blue-shifted in the 3 and 2 M GdnHCl solutions. These results indicate that the tryptophans were exposed to the hydrophilic solvent because of the fact that scFv was unfolded at GdnHCl concentrations higher than 2 M, even though the environment around tryptophans was a little changed by the decreasing of GdnHCl concentration. In all the refolding solutions, a large blue-shift to 346 nm was observed as the GdnHCl concentration changed from 2 to 1 M, and the fluorescence intensity also decreased at this same stage (data not shown). The blue-shift and accompanying decrease of intensity were also observed in the dialysates from 1 to 0.5 M GdnHCl and from 0.5 to 0 M. Decreasing the GdnHCl concentration to less than M drastically changed the environment around the scFv tryptophan residues. These results, along with the CD spectra (Fig. 3), indicate that the secondary and tertiary structures in scFv began forming at the same stage (1 M GdnHCl stage). In dialysis systems in which GSSG or L-arginine was applied, the tryptophan molecules showed emission changes similar to those observed in the simple GdnHCl solutions, except for the fact that the tryptophan emissions were more blue-shifted at the 1 and 0.5 M GdnHCl stages in the solutions with GSSG or L-arginine (Fig. 5). This result suggests that scFv folded more promptly in the GdnHCl solution with GSSG or L-arginine than it did in the simple GdnHCl solution.


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 5.   Changes in the tryptophan fluorescence emission maximum for scFv at each stage in the stepwise dialysis. Simple GdnHCl solution (solid squares), GdnHCl-GSSG solution (open triangles), GdnHCl-L-arginine solution (solid triangles), and optimal GdnHCl solution (open squares). The concentrations of scFv were adjusted to 0.5 µM.

In order to probe the hydrophobicity of scFv, we measured the fluorescence intensity of bis-ANS in the presence of scFv by adding bis-ANS to the scFv-GdnHCl solution at each stage of the dialysis (Fig. 6). The weak bis-ANS fluorescence in the 6 M GdnHCl solution was equal in intensity to the fluorescence in the scFv-free GdnHCl solution, which indicates that no hydrophobic area formed on scFv, because of its completely unfolded stage (28, 29).


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 6.   Change of bis-ANS fluorescence intensity (fluorescence maximum, ~500 nm) in the scFv solutions at each stage in the stepwise dialysis. Simple GdnHCl solution (solid squares), GdnHCl-GSSG solution (open triangles), GdnHCl-L-arginine solution (solid triangles), and optimal GdnHCl solution (open squares).

In contrast, bis-ANS became highly fluorescent as the concentration of GdnHCl was decreased to 3 and 2 M (Fig. 6). This result, which differed from the CD and tryptophan emission results (Figs. 3 and 5), indicates that a hydrophobic area on scFv was formed without discernible secondary and tertiary structures even at the high GdnHCl concentrations of 3 and 2 M. The bis-ANS fluorescence was further enhanced at the 1 M GdnHCl stage, during which the secondary and tertiary structures were forming. The hydrophobic area on scFv was most exposed to the solvent at the 0.5 M GdnHCl stage in all the refolding solutions, except for the simple GdnHCl solution, in which the bis-ANS had similar fluorescence intensity at GdnHCl concentrations of 1 and 0.5 M (Fig. 6).

Comparison of the bis-ANS result with the observation of 320-nm absorbance indicates a connection between the extent of the hydrophobic area on scFv and aggregation of scFv at the 0.5 M GdnHCl stage (Figs. 2 and 6). The hydrophobic area on scFv did not expand from the 1 M GdnHCl stage in the simple GdnHCl solution, despite the fact that the bis-ANS fluorescence intensity at the M GdnHCl stage was comparable to the intensities in the other three solutions (GdnHCl-GSSG, GdnHCl-L-arginine, and optimal GdnHCl solutions). The conformational change at the 0.5 M GdnHCl stage may be insufficient in the simple GdnHCl solution because there is nothing to stabilize the hydrophobic area. At the 0 M GdnHCl stage, at which GSSG and L-arginine were removed as well as GdnHCl, the size of the hydrophobic area on scFv rapidly decreased in all cases (Fig. 6). This result suggests that the VL and VH domains become stacked at the final stage.

Quantification of the Free Thiol Groups in scFv at Each Stage in the Stepwise Dialysis-- The free thiol groups in untreated and denatured (6 M GdnHCl) scFv were quantified at each stage by means of Ellman's assay. The pH of the denaturing solution was adjusted to 5.0 in order to prevent formation of disulfide linkage in the denaturing process. The average number of free thiol groups in scFv at the 6 M GdnHCl stage was 3.0 despite scFv having four thiol groups, indicating that a few thiol groups were naturally oxidized even in the 6 M GdnHCl solution. When the GdnHCl concentration was decreased from 6 to 3 M, the number of free thiol groups in scFv decreased from 3 to 1.5 for the untreated scFv (Fig. 7a), whereas the same number of free thiol groups was detected in the scFv denatured from the 6 and 3 M GdnHCl stages (Fig. 7b). A similar discrepancy in the number of free thiol groups at the 3 M GdnHCl stage was also observed at the 2 M GdnHCl stage, although the difference was smaller than that at the 3 M GdnHCl stage. This result reveals that the scFv structure in the 3 and 2 M GdnHCl solutions hinders DTNB access to the thiol groups, which supports the bis-ANS results suggesting that some structure was formed at the 3 and 2 M GdnHCl stages.


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 7.   Change in the amount of free thiol groups in scFv at each stage in the stepwise dialysis. a, simple GdnHCl solution; b, GdnHCl-GSSG solution; c, GdnHCl-L-arginine solution; d, optimal GdnHCl solution; untreated scFv (open circles) and scFv denatured in 6 M GdnHCl solution (solid circles).

In the dialysis from 2 M GdnHCl, oxidation of free thiol groups was significantly dependent upon the composition of the refolding solutions. In the GdnHCl solution without additives (Fig. 7a), the free thiol groups in the 2 M GdnHCl solution were spontaneously oxidized by disulfide bond formation only in the dialysis from 2 to 1 M; consequently, some free thiol groups remained even at the 0 M GdnHCl stage. These results indicate that natural oxidization cannot make a sufficient number of disulfide linkages in scFv. For the GdnHCl solutions with GSSG, Ellman's assay was used after complete exclusion of the oxidizing reagent by dialysis. The assay showed that the addition of GSSG left few free thiol groups at the 1 and 0.5 M GdnHCl stages (Fig. 7b). We attempted to detect the binding of GSSG to scFv by mass spectrometry, but the scFv without GSSG was enhanced in the GdnHCl-GSSG solution at the 1 M GdnHCl stage (data not shown). Therefore, the addition of GSSG enabled sufficient disulfide-bond formation in scFv at the 1 M GdnHCl stage.

Ellman's assay also distinguished an essentially distinct disulfide bond formation pathway in the presence of L-arginine (Fig. 7c). In the presence of L-arginine, the results differed from those observed for the simple GdnHCl and GdnHCl-GSSG solutions (Fig. 7, a and b); the thiol groups showed no disulfide bond formation behavior at the 1 M GdnHCl stage, and instead the number of thiol groups increased and reached a maximum at the 6 M GdnHCl stage (Fig. 7c). Considering the tryptophan emission results (Fig. 5), this result indicates that the folding of scFv occurred without disulfide linkage in the presence of L-arginine at the 1 M GdnHCl stage. The increase in the amount of free thiol groups might be caused by a drastic conformational change from random structure to native-like secondary and tertiary structures. Instead, the free thiol groups were naturally oxidized at the 0.5 M GdnHCl stage, but at the 0 M GdnHCl stage as many free thiol groups remained as in the simple GdnHCl solution (Fig. 7, a and c).

Disulfide bond formation in the optimal GdnHCl solution occurred by the same pathway as in the GdnHCl-GSSG solution (Fig. 7, b and d), and mass spectrometry showed enhancement of scFv without GSSG at the 1 M GdnHCl stage (data not shown). Therefore, the addition of GSSG can lead to sufficient disulfide bond formation between free thiol groups in the stepwise dialysis even in the presence of L-arginine. Although we carried out sodium lauryl sulfate-PAGE for all the refolding solutions in order to detect interchain disulfide bond formation, we observed little scFv dimer or trimer in the gel (data not shown). Furthermore, attempts at digestion with endoproteinase Lys-C detect no incorrect disulfide linkages for the scFv at each stage (data not shown). Hence, the thiol groups in scFv formed a correct intrachain linkage in all cases in this study.

Reactivity of Iodoacetic Acid with scFv at Each Stage in the Stepwise Dialysis-- In order to verify the results of the bis-ANS and Ellman's assays, we measured the MALDI-TOF mass spectra of scFv alkylated by iodoacetic acid at each stage of the dialysis. The structure of scFv prevented access of DTNB to thiol groups in the 3 and M GdnHCl solutions, and few of the thiol groups formed disulfide linkages at the 1 M GdnHCl stage in the presence of L-arginine. Fig. 8a shows the MALDI-TOF mass spectra for the scFv fragment that was alkylated by the addition of iodoacetic acid directly to the scFv solution at the 6, 3, and 2 M GdnHCl stages. To crystallize each alkylated scFv sample under the same conditions, we dried the alkylated scFv on the target after GdnHCl concentration was decreased to 2 M by dialysis. For the scFv alkylated in the 6 M GdnHCl solution, we observed unmethylated, dimethylated, and tetramethylated scFv fragments (I, II, and III in Fig. 8a-1), and the band of the dimethylated scFv was enhanced. However, the band of the unmethylated scFv was more intense at the 3 and 2 M GdnHCl stages than at the 6 M stage, and the band intensity was inversely proportional to the GdnHCl concentration at which scFv was alkylated (Fig. 8, a-2 and a-3).


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 8.   MALDI-TOF mass spectra of alkylated scFv in the simple GdnHCl solution. a-1, 6 M; a-2, 3 M; and a-3, 2 M; after denaturation from b-1, 6 M GdnHCl; b-2, 3 M GdnHCl; and b-3, 2 M GdnHCl; unmethylated fragment (I), dimethylated fragment (II), and tetramethylated fragment (III).

In contrast, the spectra of the scFv alkylated after denaturation from each GdnHCl stage by dialysis to the denaturing solution (6 M GdnHCl (pH 5.0)) were the same (Fig. 8b). In light of the results of Ellman's assay, this result can be explained by the existence of a structure around the thiol groups that hinders the access of the alkylating agent in the 3 and 2 M GdnHCl solutions.

MALDI-TOF mass spectra were also measured for scFv that had been alkylated after denaturation from the 1 and 0.5 M GdnHCl stages in the presence of L-arginine (Fig. 9). Remarkably, the spectrum for the scFv alkylated after denaturation from the 1 M GdnHCl stage containing L-arginine was identical to that from the 2 M GdnHCl stage (Fig. 9, a and b), whereas the intensity of the unmethylated scFv fragment increased at the 0.5 M GdnHCl stage (Fig. 9, b and c). This result, which is consistent with the results of Ellman's assay, indicated that most of the disulfide linkages in scFv were formed not at the 1 M GdnHCl stage but at the 0.5 M stage (Fig. 7c).


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 9.   MALDI-TOF mass spectra of alkylated scFv after denaturation from GdnHCl. a, 2 M; b, 1 M; c, 0.5 M in the GdnHCl-L-arginine solution; unmethylated fragment (I), dimethylated fragment (II), and tetramethylated fragment (III).

Structural Analysis of the Aggregated Form of scFv at the 1 and 0.5 M GdnHCl Stages-- FT-IR spectra were measured on the insoluble aggregated form of scFv at each stage (Fig. 10). The aggregated scFv in the simple GdnHCl solution at the 1 M GdnHCl stage had a broad band at around 1685 nm that is derived from the amide groups in the beta -turn structure, but a weak signal at around 1620 nm, which is assigned to the beta -strand structure, was also observed. The aggregated scFv at the 1 M GdnHCl stage had no beta -strand structure. In contrast, the aggregated scFv in the GdnHCl-GSSG solution at the 0.5 M GdnHCl stage showed relatively intense bands at around 1620 nm (Fig. 10), indicating the existence of beta -strand structure in the aggregated scFv at the 0.5 M GdnHCl stage.


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 10.   FT-IR spectra of the aggregated particles of scFv in the simple GdnHCl solution at the 1 M stage (solid line) and in the GdnHCl-GSSG solution at the 0.5 M stage (dotted line).

We also measured the spectrum of the aggregated scFv in the GdnHCl-L-arginine solution at the 0.5 M GdnHCl stage and recognized beta -strand formation as well (data not shown). Therefore, scFv formed an aggregate with native-like secondary structure when the GdnHCl concentration was reduced from 1 to 0.5 M in the GdnHCl-GSSG and GdnHCl-L-arginine solutions.

Table I shows the number of free thiol groups in the scFv aggregates at the 2, 1, 0.5, and 0 M stages. The aggregated scFv at the 1 M GdnHCl stage in the simple GdnHCl solution showed a value between those of the soluble fractions at the 2 and 1 M stages in the simple GdnHCl solution. This result indicates that the number of disulfide linkages in the aggregated scFv at the M GdnHCl stage did not provide sufficient stabilization at the GdnHCl concentration of 1 M.

                              
View this table:
[in this window]
[in a new window]
 
Table I
The number of free thiol groups, quantified by Ellman's assay, in aggregated scFv in the dialysis from 2 to 0 M GdnHCl
The values in parentheses correspond to the number of free thiol groups in the soluble fraction; no agg., no aggregation; ---, could not be measured.

At the 0.5 M GdnHCl stage, scFv formed an aggregate even in the presence of GSSG or L-arginine; however, there was a significant difference in the number of the free thiol groups in the aggregate. The aggregated scFv in the GdnHCl-GSSG solution showed the same number of thiol groups as the soluble fraction at the 1 and 0.5 M GdnHCl stages, but the number of thiol groups in the aggregate in the GdnHCl-L-arginine solution was midway between the values for the soluble fractions at the 1 and 0.5 M GdnHCl stages. These results indicate that the scFv in the GdnHCl-GSSG solution formed an aggregate independently of thiol group behavior, whereas the aggregation of scFv in the GdnHCl-L-arginine solution strongly depended on the structure of scFv without disulfide linkages. Therefore, the factors leading to the formation of aggregates at the 0.5 M GdnHCl stage in the GdnHCl-GSSG solution were essentially different from the factors in the GdnHCl-L-arginine solutions.

We also measured the sodium lauryl sulfate-PAGE without a reducing reagent for all the scFv aggregates and detected no interchain disulfide linkages (data not shown). Hence, interchain disulfide linkages, which can lead to the aggregation, were not formed even in the aggregated forms in this study.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Formation of Disulfide Linkages in the Stepwise Dialysis System-- For the folding of scFv in the stepwise dialysis system, we have provided abundant spectroscopic data on the formation of secondary and tertiary structures, hydrophobic areas, and disulfide linkages; the folding processes are illustrated in Fig. 11. When the GdnHCl concentration was reduced from 6 to 2 M, we observed a structure in which the thiol groups were inaccessible to DTNB and iodoacetic acid, even though the structure had no distinct secondary or tertiary structure (Fig. 11a).


View larger version (32K):
[in this window]
[in a new window]
 
Fig. 11.   The scFv folding pathway in the stepwise dialysis from 6 M through 3 to 2 M GdnHCl (a) and from 2 to 0 M GdnHCl (b).

In the GdnHCl solution without additives (i.e. the simple GdnHCl solution), the CD spectrum showed beta -strand formation at a GdnHCl concentration of 1 M (Fig. 3), and the formation of tertiary structure was also observed at the same stage in tryptophan fluorescence emission experiments (Fig. 5). Interestingly, most of the thiol groups in scFv were naturally oxidized at the 1 M GdnHCl stage, and no further oxidation of the residual thiol groups to disulfide linkages occurred at the stages after 1 M (Fig. 7a). The regeneration study by Goto and Hamaguchi (30) of the reduced constant fragment has demonstrated that the proximity of a pair of cysteinyl residues to each other is essential for the formation of a disulfide linkage and that the thiol groups are not easily oxidized in the interior of protein molecules.

In our study, disulfide bond formation was strongly coupled to the formation of secondary and tertiary structures and resulted from the proximity of a native pair of cysteinyl residues in a native-like intermediate that formed at the 1 M GdnHCl stage (Fig. 11b). At the stages after 1 M, steric constraints prevented oxidation of the residual thiol groups to disulfide linkages. Therefore, the introduction of thiol-disulfide interchange reaction by GSSG at the 1 M GdnHCl stage was best suited for making the correct disulfide linkages since each native pair of cysteinyl residues in the VH and VL domains was in close proximity to each other at this stage.

The importance of the disulfide linkage in the immunoglobulin fold has been demonstrated in a large number of studies (2, 10, 19), and recent studies (31-33) on disulfide-free antibody suggest that the stabilization of the disulfide-free antibodies requires amino acid exchange to compensate for the loss of folding stability upon cleavage of the disulfide linkage, although a functional disulfide-free scFv can be obtained. A mutation study on the scFv of the antibody hu4D5-8 against the extracellular domain of human epidermal growth factor receptor-2 indicates that the wild-type 4D5 scFv with a disulfide linkage in each of VH and VL domains has a higher midpoint of denaturation in the equilibrium transition than the variants lacking one or two disulfide linkages (10), which means that the 4D5 scFv with the native disulfide linkages is folded at a higher GdnHCl concentration than the variants.

In our study, the trough derived from the beta -strand structure that was observed in the CD spectrum in the presence of GSSG at the 1 M GdnHCl stage was more distinct than that observed in the CD spectrum of the simple GdnHCl solution (Fig. 4). The change in the tryptophan fluorescence emission maximum also showed more scFv folding in the GdnHCl-GSSG solution than in the simple GdnHCl solution (Fig. 5). The sufficient disulfide bond formation in scFv caused by the addition of GSSG accelerated the formation of the immunoglobulin fold (Fig. 11b). In the simple GdnHCl solution at the 1 M GdnHCl stage, scFv formed an aggregate with more free thiol groups than in the soluble scFv, whereas no aggregated scFv was formed in the GdnHCl-GSSG solution at the same stage (Table I).

Ramm et al. (10) have reported that the 4D5 scFv variant with no disulfide linkage in either domain (V<UP><SUB>L</SUB><SUP>−</SUP></UP>V<UP><SUB>H</SUB><SUP>−</SUP></UP>) is prone to aggregation and, furthermore, that the 4D5 scFv variant with a disulfide linkage in VL but not in VH (V<UP><SUB>L</SUB><SUP>+</SUP></UP>V<UP><SUB>L</SUB><SUP>−</SUP></UP>) is less stable than the V<UP><SUB>L</SUB><SUP>−</SUP></UP>V<UP><SUB>H</SUB><SUP>−</SUP></UP> variant. Therefore, in our study, the lack of aggregation of the HyHEL-10 scFv at the 1 M stage upon the addition of GSSG was due to the immediate formation of native disulfide linkages; the simultaneous formation of the disulfide linkages in both domains might also be a factor in suppressing aggregation at the 1 M GdnHCl stage. Possible factors leading to aggregation in the folding of scFv could be inappropriate disulfide-linkage formation and non-native association of the constituent polypeptide chains. Our results suggest that the native disulfide linkages inhibit non-native association of the constituent polypeptide chains at the 1 M GdnHCl stage. In the simple GdnHCl solution, the slow formation of the disulfide linkages by natural oxidation allowed non-native association, which led to the formation of aggregated particles (Fig. 11b). The difference between the rate of disulfide bond formation in the VL domain and that in the VH domain might also lead to aggregation at the 1 M GdnHCl stage.

Effect of L-Arginine on the Folding of scFv in the Stepwise Dialysis System-- In the GdnHCl-GSSG solution, the essential factor that suppressed aggregation of scFv at the 1 M GdnHCl stage was the immediate formation of native disulfide linkages by GSSG, linkages that stabilized a native-like intermediate state (Fig. 11b). In contrast, few of the disulfide linkages in the GdnHCl-L-arginine solution were formed at the 1 M GdnHCl stage, despite the fact that there was no aggregation of scFv (Figs. 2 and 7c). Although the CD spectra of scFv could not be measured in the presence of L-arginine, because of the strong CD activity of L-arginine, the changes in the tryptophan fluorescence emission spectra in the dialysis system showed that the scFv with a partially folded structure but no disulfide linkages was stabilized at the 1 M GdnHCl stage by L-arginine (Fig. 11b).

L-Arginine has been used in many refolding systems to suppress protein aggregation (19, 34-37), and the reagent is considered to be a labilizing agent that facilitates correct folding by destabilizing incorrectly folded structures (19, 33-35). However, the theoretical basis for the mechanism by which L-arginine suppresses aggregation remains undetermined.

In the simple GdnHCl solution without GSSG or L-arginine, the factor that led to scFv aggregation at the 1 M GdnHCl stage was non-native association of the polypeptide chains, which would be inhibited by immediate disulfide bond formation in the VL and VH domains. Considering that scFv was partially folded but had no disulfide linkages and that the hydrophobic area on scFv increased in size at the 1 M GdnHCl stage in the GdnHCl-L-arginine solution (Figs. 5, 6, and 7c), the addition of L-arginine may have inhibited association between the partially folded elements so that the thiol groups were unable to approach each other (Fig. 11b).

At the 0.5 M GdnHCl stage, in which exposure of the hydrophobic area on scFv was at a maximum (Fig. 5), the aggregation of scFv in the GdnHCl-GSSG solution could be suppressed by the addition of L-arginine (Fig. 2). This result supports the proposal that the association between the partially folded elements was inhibited in the presence of L-arginine, since the correct disulfide linkages in scFv had already been formed by GSSG at the 1 M GdnHCl stage (Fig. 7, c and d). In contrast, scFv was aggregated at the 0.5 M GdnHCl stage in the GdnHCl-L-arginine solution without GSSG. At this stage, most of the thiol groups in the soluble scFv were naturally but not completely oxidized to disulfide, and furthermore, some free thiol groups remained in the aggregated scFv, as in the case of the folding in the simple GdnHCl solution (Table I). This thiol group behavior implies that the native pair of cysteinyl residues approached each other at the 0.5 M GdnHCl stage in the GdnHCl-L-arginine solution but that slow disulfide-bond formation by natural oxidation led scFv to form aggregates (Fig. 11b). Ahn et al. (36) have suggested that L-arginine supplements the role of GdnHCl because they found that the optimal GdnHCl concentration for refolding decreased when L-arginine was added. Therefore, the combination of GdnHCl and L-arginine may be necessary for inhibition of the association between the partially folded elements. Without the formation of native disulfide linkages, the association in the scFv could not be inhibited even by the combination of 0.5 M GdnHCl and 0.4 M L-arginine. Attempts to elucidate the mechanism by which the addition of L-arginine inhibits the association between the partially folded elements are in progress.

Highly Efficient Refolding of scFv in the Optimal Stepwise Dialysis System-- We achieved a high refolding yield with our stepwise dialysis system by adding GSSG and L-arginine at the appropriate stages (the 1 and 0.5 M GdnHCl stages, respectively). Our spectroscopic studies emphasize that the addition of an oxidizing reagent is necessary at the stage in which the native pairs of cysteinyl residues approach each other, because of the formation of secondary and tertiary structures (at the 1 M GdnHCl stage). In our previous attempts (23), the addition of GSSG at the 1 M GdnHCl stage resulted in complete recovery of the biological activity of the soluble scFv at the 0 M GdnHCl stage, whereas adding GSSG at other times resulted in recoveries of less than 60%. From our present study, we can interpret the reduced recovery of activity as being the result of steric constraints that prevented GSSG from accessing the thiol groups. In the optimal GdnHCl solution, GSSG and L-arginine were added simultaneously at the 1 and 0.5 M GdnHCl stages. Considering that the disulfide bond formation pathway in the optimal GdnHCl solution was similar to that in the GdnHCl-GSSG solution (rather than to that in the GdnHCl-L-arginine solution), the effect of GSSG was more immediate than that of L-arginine. Therefore, the formation of native disulfide linkages at the 1 M GdnHCl stage by the addition of GSSG and inhibition of the association between the partially folded elements by the presence of L-arginine at the 0.5 M stage enabled complete recovery of biological activity and no aggregation in the optimal stepwise dialysis system. In addition, the current study shows that the presence of L-arginine delayed disulfide bond formation (Fig. 7c), which suggests that we should introduce GSSG at the 0.5 M GdnHCl stage if L-arginine is already contained in the refolding buffer. Hence, the appropriate stage for adding GSSG is not necessarily the same as that for adding L-arginine; however, the timings of the introduction of GSSG and L-arginine are strongly coupled each other.

Recently, we succeeded in approving the refolding efficiency for interleukin-21 by the introduction of reducing reagent (GSH) at the 1 and 0.5 M GdnHCl stages, which induces thiol-disulfide interchange reaction (38), which is different from the result of scFv that the introduction of reducing reagent decreases the refolding efficiency (23). Considering that interleukin-21 has an alpha -helix as a secondary structure (38), the possibility that the addition of GSH in the stepwise dialysis system improves the yield of correct folding is strongly dependent upon the native structure. The refolding process with thiol-disulfide interchange reaction in the stepwise dialysis system will be reported in the future.

Conclusion-- Various spectroscopic data were used to elucidate the folding process of scFv from the denatured and reduced state in the stepwise dialysis system. Disulfide bond formation occurred at the particular stage when the secondary and tertiary structures were formed, and the formation of correct disulfide linkages stabilized the native-like intermediate at the same stage. The addition of GSSG at that stage was most effective for making the native disulfide linkage because a native pair of cysteinyl residues were in proximity to each other in the native-like intermediate. L-Arginine may have inhibited association between the constituent polypeptide chains or the partially folded elements in scFv but that inhibition depended on the combination of GdnHCl and L-arginine. The combination of 1 M GdnHCl and 0.4 M L-arginine was especially good for stabilizing the partially folded intermediate with reduced thiol groups, and as a result, the free thiol groups formed disulfide linkages through a different pathway in the GdnHCl-L-arginine solution than in the GdnHCl-GSSG solution. The highly efficient refolding of scFv in the presence of GSSG and L-arginine (the optimal GdnHCl solution system) was achieved by the formation of native disulfide linkages at the 1 M GdnHCl stage and inhibition of association between the partially folded elements at the 0.5 M stage. In general, the refolding of scFv is carried out by dilution of the denatured protein in a refolding buffer with a redox reagent (GSH/GSSG) and L-arginine, and the timing of the addition of these additives has not been controlled. In this study, however, we demonstrated the importance of controlling the additive effects and correctly timing their introductions. We have found that applying the optimal stepwise dialysis system is also effective for other immunoglobulin-folded proteins such as Fab and various receptors. This work will be reported in the future.

    FOOTNOTES

* This work was supported by a grant-in-aid for the Original Industrial and Technology R & D Promotion Program from the New Energy and Industrial Technology Development Organization of Japan and by a Scientific Research Grant 14780502 from the Ministry of Education, Science, Sports, and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence may be addressed. Tel./Fax: 81-22-217-7276; E-mail: tsumoto@mail.cc.tohoku.ac.jp.

|| To whom correspondence may be addressed. Tel.: 81-22-217-7274; Fax: 81-22-217-6164; E-mail: kmiz@mail.cc.tohoku.ac.jp.

Published, JBC Papers in Press, January 7, 2003, DOI 10.1074/jbc.M212247200

    ABBREVIATIONS

The abbreviations used are: CL, light chain of immunoglobulin constant regions; bis-ANS, 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid dipotassium salt; beta -ME, 2-mercaptoethanol; DTNB, 5,5'-dithiobis(2-nitrobenzoic acid); FT-IR, Fourier transformed-infrared; Fv, fragment of immunoglobulin variable regions; GdnHCl, guanidine hydrochloride; HyHEL-10, mouse anti-lysozyme monoclonal antibody; MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight; scFv, single-chain Fv fragment; VH, heavy chain domain in Fv; VL, light chain in Fv.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Goto, Y., and Hamaguchi, K. (1979) J. Biochem. (Tokyo) 86, 1433-1441[Abstract]
2. Glockshuber, R., Schmidt, T., and Plückthun, A. (1992) Biochemistry 31, 1270-1279[Medline] [Order article via Infotrieve]
3. Alzari, P. M., Lascombe, M. B., and Poljak, R. J. (1988) Annu. Rev. Immunol. 6, 555-580[CrossRef][Medline] [Order article via Infotrieve]
4. Hunkapiller, T., and Hood, L. (1989) Adv. Immunol. 44, 1-63[Medline] [Order article via Infotrieve]
5. Davies, D. R., Padlan, E. A., and Sherriff, S. (1990) Annu. Rev. Biochem. 59, 439-473[CrossRef][Medline] [Order article via Infotrieve]
6. Amzel, L. M., and Poljak, R. J. (1979) Annu. Rev. Biochem. 48, 961-997[CrossRef][Medline] [Order article via Infotrieve]
7. Goto, Y., and Hamaguchi, K. (1982) J. Mol. Biol. 156, 891-910[Medline] [Order article via Infotrieve]
8. Proba, K., Honegger, A., and Plückthun, A. (1997) J. Mol. Biol. 265, 161-172[CrossRef][Medline] [Order article via Infotrieve]
9. Langedijk, A. C., Honegger, A., Maat, J., Planta, R. J., van Schaik, R. C., and Plückthun, A. (1998) J. Mol. Biol. 283, 95-110[CrossRef][Medline] [Order article via Infotrieve]
10. Ramm, K., Gehrig, P., and Plückthun, A. (1999) J. Mol. Biol. 290, 535-546[CrossRef][Medline] [Order article via Infotrieve]
11. Jäger, M., Gehrig, P., and Plückthun, A. (2001) J. Mol. Biol. 305, 1111-1129[CrossRef][Medline] [Order article via Infotrieve]
12. Carter, P., and Merchant, A. M. (1997) Curr. Opin. Biotechnol. 8, 449-454[CrossRef][Medline] [Order article via Infotrieve]
13. Holt, L. J., Enever, C., de Wildt, R. M., and Tomlinson, I. M. (2000) Curr. Opin. Biotechnol. 11, 445-449[CrossRef][Medline] [Order article via Infotrieve]
14. Plückthun, A. (1992) Immunol. Rev. 130, 151-188[Medline] [Order article via Infotrieve]
15. Skerra, A. (1993) Curr. Opin. Immunol. 5, 256-262[CrossRef][Medline] [Order article via Infotrieve]
16. Field, H., Yarranton, G. T., and Rees, A. R. (1990) Protein Eng. 3, 641-647[Medline] [Order article via Infotrieve]
17. Tsumoto, K., Ogasahara, K., Ueda, Y., Watanabe, K., Yutani, K., and Kumagai, I. (1995) J. Biol. Chem. 270, 18551-18557[Abstract/Free Full Text]
18. Jurado, P., Ritz, D., Beckwith, J., de Lorenzo, V., and Fernández, L. A. (2002) J. Mol. Biol. 320, 1-10[CrossRef][Medline] [Order article via Infotrieve]
19. Buchner, J., and Rudolph, R. (1991) Biotechnology 9, 157-162[Medline] [Order article via Infotrieve]
20. Huston, J. S., Levinson, D., Mudgett-Hunter, M., Tai, M.-S., Novotny, J., Margolies, M. N., Ridge, R. J., Bruccoleri, R. E., Haber, E., Crea, R., and Oppermann, H. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 5879-5883[Abstract]
21. Pantoliano, M. W., Bird, R. E., Johnson, S., Asel, E. D., Dodd, S. W., Wood, J. F., and Hardman, K. D. (1991) Biochemistry 30, 10117-10125[Medline] [Order article via Infotrieve]
22. Cheadle, C., Hook, L. E., Givol, D., and Ricca, G. A. (1992) Mol. Immunol. 29, 21-30[CrossRef][Medline] [Order article via Infotrieve]
23. Tsumoto, K., Shinoki, K., Kondo, H., Uchikawa, M., Juji, T., and Kumagai, I. (1998) J. Immunol. Methods 219, 119-129[CrossRef][Medline] [Order article via Infotrieve]
24. Tsumoto, K., Nakaoki, Y., Ueda, Y., Ogasahara, K., Yutani, K., Watanabe, K., and Kumagai, I. (1994) Biochem. Biophys. Res. Commun. 201, 546-551[CrossRef][Medline] [Order article via Infotrieve]
25. Uchiyama, H., Perez-Prat, E. M., Watanabe, K., Kumagai, I., and Kuwajima, K. (1995) Protein Eng. 8, 1153-1161[Medline] [Order article via Infotrieve]
26. Sedlak, J., and Lindsay, R. H. (1968) Anal. Biochem. 25, 192-205[Medline] [Order article via Infotrieve]
27. Kondo, H., Shiroishi, M., Matsushima, M., Tsumoto, K., and Kumagai, I. (1999) J. Biol. Chem. 274, 27623-27631[Abstract/Free Full Text]
28. Goto, Y., and Fink, A. L. (1989) Biochemistry 28, 945-952[Medline] [Order article via Infotrieve]
29. Chapeaurouge, A., Johansson, J. S., and Ferreira, S. T. (2001) J. Biol. Chem. 276, 14861-14866[Abstract/Free Full Text]
30. Goto, Y., and Hamaguchi, K. (1981) J. Mol. Biol. 146, 321-340[Medline] [Order article via Infotrieve]
31. Frisch, C., Kolmar, H., Schmidt, A., Kleemann, G., Reinhardt, A., Pohl, E., Usón, I., Schneider, T. R., and Fritz, H.-J. (1996) Folding Des. 1, 431-440[Medline] [Order article via Infotrieve]
32. Proba, K., Wörn, A., Honegger, A., and Plückthun, A. (1998) J. Mol. Biol. 275, 245-253[CrossRef][Medline] [Order article via Infotrieve]
33. Wörn, A., and Plückthun, A. (1998) FEBS Lett. 427, 357-361[CrossRef][Medline] [Order article via Infotrieve]
34. Orsini, G., and Goldberg, M. E. (1978) J. Biol. Chem. 253, 3453-3458[Abstract]
35. Jaenicke, R., and Rudolph, R. (1989) in Protein Structure: A Practical Approach (Creighton, C. R., ed) , pp. 191-223, IRL Press at Oxford University Press, Oxford
36. Ahn, J. H., Lee, Y. P., and Rhee, J. S. (1997) J. Biotechnol. 54, 151-160[CrossRef][Medline] [Order article via Infotrieve]
37. Qiao, Z.-S., Guo, Z.-Y., and Feng, Y.-M. (2001) Biochemistry 40, 2662-2668[CrossRef][Medline] [Order article via Infotrieve]
38. Asano, R., Kudo, T., Makabe, K., Tsumoto, K., and Kumagai, I. (2002) FEBS Lett. 528, 70-76[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.