From the 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
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ABSTRACT |
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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.
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 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.
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- 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
( 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).
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 2 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.
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
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 2 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.
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).
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 1 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.
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 2 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).
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).
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
We also measured the spectrum of the aggregated scFv in the
GdnHCl-L-arginine solution at the 0.5 M GdnHCl
stage and recognized
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 1 M GdnHCl stage did not
provide sufficient stabilization at the GdnHCl concentration of 1 M.
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.
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).
In the GdnHCl solution without additives (i.e. the simple
GdnHCl solution), the CD spectrum showed
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
Ramm et al. (10) have reported that the 4D5 scFv variant
with no disulfide linkage in either domain
(V 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 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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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 6 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).
-ME) at a 50-fold molar excess relative to the protein. After
-ME was removed by dialysis against the same Tris-HCl buffer without
-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.
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Fig. 1.
The stepwise dialysis system.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (12K):
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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).
-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
-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
-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.
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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.
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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).
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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.
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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).
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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).
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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).
View larger version (15K):
[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).
-turn
structure, but a weak signal at around 1620 nm, which is assigned to
the
-strand structure, was also observed. The aggregated scFv at the
1 M GdnHCl stage had no
-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
-strand structure in
the aggregated scFv at the 0.5 M GdnHCl stage.
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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).
-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.
The number of free thiol groups, quantified by Ellman's assay, in
aggregated scFv in the dialysis from 2 to 0 M GdnHCl
, could not be
measured.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (32K):
[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).
-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.
-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).
-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.
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FOOTNOTES |
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* 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
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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;
-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.
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---|
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 |
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 |
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 |
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] |