1 Department of Biotechnology, Royal Institute of Technology (KTH), Stockholm Centre for Physics, Astronomy and Biotechnology (SCFAB),SE-106 91 Stockholm and 2 Affibody AB, Box 20137, SE-161 02 Bromma, Sweden
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Abstract |
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Keywords: affinity chromatography/deamidation/protein G/purification/stabilization
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Introduction |
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Asparagine residues have been recognized as the major contributors to the alkaline sensitivity, but also glutamine and to a lesser extent aspartate residues can be chemically modified (Stephenson and Clarke, 1989; Wright, 1991
; Tomizawa et al., 1995
). These modifications may change the function or the potency of a protein or a peptide. The reaction mechanism involves the main chain peptide nitrogen of the succeeding residue, which functions as the nucleophile and attacks the side chain carbonyl of the asparagine, glutamine or aspartate. This cyclization reaction results in a cyclic imide intermediate, which is hydrolyzed to isomerization products (aspartate/glutamate and isoaspartate/isoglutamate). The resulting isomers may be found in their L- or D-form. In addition, cleavage of the peptide bond can occur. These reactions are spontaneous but the rate is severely increased at alkaline pH. Also, temperature and ionic strength are parameters that influence the rate (Geiger and Clarke, 1987
). Different proteins are modified to a different extent since the deamidation/isomerization rate is highly sequence and conformation dependent (Kossiakoff, 1988
; Lura and Schirch, 1988
; Wearne and Creighton, 1989
).
In this paper, we evaluate if the protein engineering strategy used to stabilize the albumin-binding domain (ABD) derived from streptococcal protein G (SPG) (Gülich et al., 2000b) can be used to increase the stability towards alkaline treatment for yet another protein ligand, the C2 domain. This is a well known and thoroughly characterized protein, exploited in commercially available protein-based affinity chromatography media. The C2 domain is derived from SPG, a cell-surface protein from Streptococcus G148 (Olsson et al., 1987
), and has the potential to be used in large-scale purification of monoclonal antibodies. Streptococcal protein G is a multidomain protein with separated albumin-binding and immunoglobulin-binding regions (Nygren et al., 1988
). SPG exhibits a broad spectrum of binding to IgG subclasses, and binds to all four human subclasses of IgG, as well as several animal IgG subclasses (Björck and Kronvall, 1984
; Åkerström et al., 1985
; Olsson et al., 1987
). The IgG-binding region consists of three domains, C1, C2 and C3, exhibiting high sequence homology with only a few residue exceptions (Figure 1A
). The C2 domain is a small protein constituted of only 55 residues. We have chosen to produce it with an additional leader and linker sequence of 13 amino acids derived from the leader region in the TrpLE expression vector (asparagines excluded) (Figure 1A
). The structure of C2 and the homologous C1 and C3 domains has been determined both alone and in complex with Fab and Fc by NMR (Gronenborn et al., 1991
; Lian et al., 1992
) and X-ray crystallography (Achari et al., 1992
; Derrick and Wigley, 1992
, 1994
; Gallagher et al., 1994
; Sauer-Eriksson et al., 1995
). It is constituted of a four-stranded ß-sheet crossed diagonally by a single
-helix. The central two strands comprising the N- and C-termini are parallel. The outer two strands are anti-parallel to the central two strands and connected by the helix (Figure 1B
). The C2 domain has no additional stabilizing features such as disulfide bridges or bound prosthetic groups. The topology, the extensive H-bonding network, and the tightly packed hydrophobic core are probably responsible for the high thermal stability. Almost all residues are involved in regular secondary structure elements, which ensure a large number of stabilizing H-bonds (Gronenborn et al., 1991
). The domain shows strong interaction to the Fc fragment (Sauer-Eriksson et al., 1995
) and also weak interaction to the Fab fragment (Derrick and Wigley, 1992
, 1994
) of IgG from various species including human. Hence, these characteristics have given the IgG-binding region of SPG a biochemical utility in purifying antibodies, as well as in folding and stability studies.
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Materials and methods |
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The plasmid pC2C3 (Nygren et al., 1988) was used as template in a site-directed mutagenesis using a two-step PCR technique (Higuchi et al., 1988
). Oligonucleotides coding for the 13-residue leader and linker sequence of the TrpLE expression vector (asparagines excluded), the nine-residue linker sequence in the dimers, and the different residue substitutions were synthesized by Interactiva (Interactiva Biotechnologie GmbH, Ulm, Germany). The cloning was performed essentially as described by Sambrook et al. (Sambrook et al., 1989
), using the restriction enzymes XbaI and HindIII (MBI Fermentas Inc., Amhurst, NY), and the vector pDHZ (Jansson et al., 1996
). The sequence of inserted fragments was verified on a MegaBACETM 1000 DNA Sequencing System, using cycle sequencing based on the dideoxy method (Sanger et al., 1977
) with MegaBACETM terminator chemistry according to the suppliers recommendations (Amersham Biosciences, Uppsala, Sweden). Escherichia coli strain RR1
M15 (American Type Culture Collection, Rockville, MA) was used during cloning procedures and 017 (Olsson and Isaksson, 1979
) was used for expression of the different gene products.
Production and purification
The wild-type protein and mutants thereof were produced according to the protocol described by Gülich et al. (Gülich et al., 2000a). After 20 h of cultivation the cells were harvested by centrifugation (10 min at 5000 g). The cells were resuspended in 25 ml of cold TST (25 mM TrisHCl pH 7.5, 150 mM NaCl, 1.25 mM EDTA, 0.05% Tween 20) followed by sonication by VibraTM cell (Sonics and Materials Inc., Danbury, CT). The soluble gene products were purified as described by Gülich et al. (Gülich et al., 2000a
). The bound material was eluted with 0.5 M HAc pH 2.9. The amount of protein was estimated by absorbance measurements at 280 nm using the specific absorbance coefficient, a (l g-1 cm-1) (Gill and von Hippel, 1989
), (C2 1.244; C2N7A, C2N34A, C2N36A 1.251; C2N7,36A 1.258; C2N7,36AD35E, C2N7,36AD39E 1.255; C2N7,36AD21,45,46E 1.251; C2N7,36AQ31A 1.267; C2dim 1.385; C2N7,36Adim 1.402; C2cys 1.227; C2dimcys 1.376; C2N7,36A-dimcys 1.392). The concentration was confirmed by analysis of amino acid composition in a Beckman 6300 amino acid analyzer (Beckman, Fullerton, CA) after hydrolysis in 6 M HCl at 155°C for 45 min.
The homogeneity and the molecular weights were confirmed by mass spectrometry using a mass spectrometer (Micromass, Manchester, UK) equipped with an electrospray unit. Calculated and detected molecular weights of the main peak: C2 7663.4 (7663); C2N7A, C2N34A, C2N36A 7620.4 (7620); C2N7,36A 7577.4 (7577); C2N7,36AD35E, C2N7,36AD39E 7591.4 (7591); C2N7,36AD21,45,46E 7619.4 (7619); C2N7,36AQ31A 7520.3 (7520); C2dim 14681.1 (14681); C2N7,36Adim 14509.0 (14509); C2cys 7766.6; C2dimcys 14784.3; C2N7,36Adimcys 14612.2.
Biospecific interaction analysis
A BiacoreTM 2000 instrument (Biacore, Uppsala, Sweden) was used to analyze the affinity of the different variants to the Fc fragment of IgG. Human polyclonal Fc (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) was immobilized on the carboxylated dextran layer of a CM5 sensor chip (research grade) (Biacore) by utilizing the N-hydroxysuccinimide and N-ethyl-N'-(3-diethylaminopropyl)-carbodiimide chemistry. Immobilization of Fc resulted in 1500 RU. Six different concentrations (0.10.6 µM) of C2 and mutants thereof were prepared in HBS (10 mM HEPES, 0.15 M NaCl, 3.4 mM EDTA, 0.005% surfactant P20, pH 7.4) and injected as duplicates in random order at a flow rate of 30 µl/min. Five millimolar HCl was used to regenerate the surface. The BIA evaluation 3.0.2b software (Biacore) was used to evaluate the data employing a 1:1 (Langmuir) model. This model is an approximation for the interaction but since all variants are evaluated using the same parameters, the kinetic constants are comparable. The signals from a blank surface were subtracted from the Fc surface and apparent ka (M-1 s-1), kd (s-1) and KA(M-1) were calculated.
The free thiol group of the C-terminal cysteine in C2cys, C2dimcys and C2N7,36Adimcys was used for directed immobilization to a CM5 sensor chip (Biacore) pre-activated with NHS/EDC chemistry according to the suppliers recommendations. 2-(2pyridinyldithio)ethaneamine hydrochloride was injected over the surfaces prior to immobilization of the ligands, which were reduced in 10 mM dithiothreitol followed by desalting on a SephadexTM G-25 column (Amersham Biosciences) into 30 mM NH4Ac pH 4.0. The immobilization resulted in 900 RU of each ligand. Human polyclonal Fc and Fab fragments (0.1 and 0.3 µM) (Jackson ImmunoResearch Laboratories Inc.) in HBS buffer were injected as duplicates in random order at a flow rate of 30 µl/min. Ten millimolar HCl was used for regeneration. The signals from a blank surface were subtracted from the ligand surfaces.
Affinity chromatography with a CIP protocol
C2 and mutants thereof were covalently coupled to HiTrapTM columns (Amersham Biosciences) by using the N-hydroxysuccinimide chemistry according to the manufacturers recommendations. An ordinary affinity chromatography scheme was followed on the ÄKTATM Explorer 10 (Amersham Biosciences). Human polyclonal IgG in TST was loaded on the columns. The columns were washed with TST and 5 mM NH4Ac pH 5.5 before elution with 0.5 M HAc pH 2.8. The eluted material was detected at 280 nm. NaOH (0.1 M) was used as cleaning agent between the different cycles. Each pulse with NaOH was 2 min.
Circular dichroism spectroscopy
A J-720 spectropolarimeter (JASCO, Tokyo, Japan) was used to analyze the secondary structure content. Samples of 0.1 mg/ml were prepared in 10 mM K3PO4 pH 6.0. Spectra were recorded in the far UV region from 250 to 195 nm at 20°C in a quartz cell of pathlength 0.1 cm, and with a scan speed of 20 nm/min. Each spectrum was the mean of three accumulated scans and the final spectra were converted into mean residue ellipticity (deg cm2 dmol-1).
Thermal denaturation of the different variants was monitored by following the CD signal at 217 nm as a function of temperature. Samples of 0.05 mg/ml were prepared in 10 mM NaAc pH 5.3. This pH was chosen since previous studies of the homologous C1 and C3 domains suggest that the protein is most stable at this pH. However, native structure is maintained at 25°C over a wide pH range (pH 1.511.0) (Alexander et al., 1992a,b
). The CD signals were measured in a 1-cm quartz cell. The interval between measure points was 5°C. Signals were transformed to apparent fraction of unfolded protein, Fapp =(yf - yobs) / (yf - yu), where yf is the value of the folded state, yobs is the observed value at different temperatures and yu is the value of the unfolded state, and plotted versus the temperature. Tm
was read from the graph at Fapp = 0.5. The temperature was also lowered in order to investigate reversibility of the denaturation.
Chemical denaturation was also monitored by following the signal at 217 nm as a function of the concentration of denaturant. Samples of 0.25 mg/ml were prepared in 10 mM NaAc pH 5.3. A stock solution of 8 M GdnHCl (Sigma Chemical Co., St. Louis, MO) was mixed with the protein solution to achieve different concentrations of denaturant. Measurements took place in a 0.1-cm quartz cell. Signals were transformed to Fapp values and plotted versus the concentration of denaturant. Cm was read from the graph at Fapp = 0.5.
RP-HPLC analysis after NaOH incubation
Proteins incubated in NaOH were also analyzed by RP-HPLC. After different incubation times in 0.1 M NaOH at room temperature (RT) the samples were injected directly onto a SourceTM 5RPC ST4.6/150 column (Amersham Biosciences) suited for high pH. The apparatus was a Hewlett Packard Series II 1090 Liquid Chromatograph (Wilmington, DE). A linear gradient from 30 to 50% of acetonitrile with 0.25% pentafluoropropionic acid for 20 min was used. The peaks were monitored at 280 nm. The eluted peaks were collected and analyzed on a VG Platform mass spectrometer (Micromass) equipped with an electrospray unit.
Isoelectric focusing gel electrophoresis (IEF)
The sensitivity towards deamidation for C2 and C2N7,36A was also investigated using IEF under non-denaturing conditions. The proteins were incubated in 0.1 M NaOH for 2 h at RT. The buffer was changed by gel filtration using SephadexTM G-25 columns (Amersham Biosciences) equilibrated with milliQ-water. The IEF gel was casted and run on a Model 111 Mini IEF cell (Bio-Rad Laboratories, Hercules, CA) according to the manufacturers recommendations using AmpholineTM preblended pH 4.06.5 (Amersham Biosciences). The IEF calibration kit was purchased from Amersham Biosciences.
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Results |
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Biospecific interaction analysis of the asparagine mutants
The surface plasmon resonance (SPR) technology was used to investigate the interaction between the Fc fragment of IgG and the C2 variants. This technology gives information on affinities as well as the kinetics of both association and dissociation in real time. Hence, it is well suited for detailed screening of the interactions of modified proteins. The sensorgrams reveal a similar binding behavior for C2, C2N7A and C2N36A (Figure 2A). C2 and C2N7A show very similar kinetic parameters (Table I
). This can be expected since Asn7 is situated on the other side of the domain facing away from the Fc-interacting region. The C2N36A variant has a slightly decreased dissociation rate constant indicating an increased stability of the complex with Fc (Table I
). The C2N34A mutant shows a very weak interaction with the Fc fragment and no reliable kinetic parameters could be calculated (Figure 2A
). Based on these data a double mutant was designed, C2N7,36A, preserving Asn34 in the sequence. The kinetic parameters of C2N7,36A, were shown to be very similar to that of C2N36A. Also, a triple mutant with all three asparagines substituted for alanines was designed for characterization. Interestingly, we were unable to produce this triple mutant in E.coli cells. Therefore, a gene fusion strategy was employed, taking advantage of the positive characteristics of the ABD (Kraulis et al., 1996
). The idea was to render an affinity purification procedure (Ståhl et al., 1999
) possible by fusing the ABD domain C-terminally to C2N7,34,36A. However, this strategy did not result in any product, thus indicating that substitution of all asparagines results in a very labile, probably incorrectly folded protein.
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Affinity chromatography with an integrated CIP step
The ability of C2 and all mutated variants thereof to function as affinity ligands in an ordinary purification scheme for IgG antibodies was investigated by immobilization of the different domains on HiTrapTM columns (Amersham Biosciences). Between the different cycles, a 2-min CIP step consisting of 0.1 M NaOH was integrated. The column with immobilized wild-type protein showed a fast decrease in capacity. After a total of 18 min of exposure towards NaOH the C2 column has only 30% capacity left. The C2N7A column reveals a similar degradation pattern. The C2N34A column loses capacity even faster than wild-type (Figure 3
). This column also shows much lower initial binding capacity probably because the Asn34 is involved in the Fc interaction. However, the C2N36A column shows a remarkable increase of stability. After 28 min of exposure this variant still exhibits almost 90% capacity. The double mutant C2N7,36A shows a similar behavior (Figure 3
).
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Also, the dimer of the double mutant C2N7,36A exhibited improved stability towards alkaline conditions compared to the wild-type protein, indicating that the linker region used is not degraded during the CIP step. This agrees with the fact that the linker does not include any asparagines, aspartates or glutamines.
Circular dichroism spectroscopy
The secondary structure content of C2 and the different mutated variants was compared using CD spectroscopy (Johnson, 1990; Schmid, 1997
). This technology is suited to detect secondary structure changes in proteins. A protein constituted of all
-helix structure exhibits minima at 208 and 222 nm and a maximum at
192 nm. Similarly, a protein constituted of ß-sheet structure exhibits a minimum at 217 nm and a maximum at
198 nm (Schmid, 1997
). The secondary structure of C2 represents a mixture of
-helix and ß-sheet components, but the ß-sheet structure predominates. As can be seen from the far-UV CD spectra, all variants show a typical spectrum of a protein containing only a moderate amount of
-helix structure. Hence, all variants exhibit the characteristic parental protein G fold. The CD signal is only slightly decreased for some of the variants compared to wild-type C2, indicating only small changes in the secondary structure upon mutation (Figure 4
).
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RP-HPLC analysis after NaOH incubation
RP-HPLC analysis was also used to detect degradation of C2 and C2N7,36A during NaOH incubation. After 2 h of incubation in 0.1 M NaOH the original peak of the wild-type protein totally disappeared (data not shown). In contrast to wild-type, the double mutant still shows a peak (slightly decreased) and mass spectrometry analysis reveals the same molecular weight as untreated protein, 7577 Da (data not shown).
Isoelectric focusing gel electrophoresis
The deamidation event is readily detected using IEF since the asparagines and glutamines are modified to aspartate and glutamate residues, respectively. This results in an increased negative charge of the protein. The gel can be run under non-denaturating conditions since all relevant residues are surface exposed. The theoretical pI of C2 and C2N7,36A is 4.66 and for each deamidated asparagine or glutamine a decrease of 0.12 pI units should be expected. Observed pI is 4.55 for untreated C2 and the double mutant (Figure 6). After 2 h of incubation in 0.1 M NaOH the C2 protein reveals a total transformation to a new more negatively charged species (Figure 6
). The double mutant on the other hand reveals no additional charged species indicating that the deamidation-susceptible residue is substituted (Figure 6
).
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Discussion |
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The biospecific interaction analysis revealed that the two single mutations N7A and N36A resulted in constructs with retained affinity to Fc (Figure 2A; Table I
). However, a significant decrease in affinity to IgG was observed when substituting Asn34 (Figure 2A
). This is rather predictable since Asn34 is one of the most important amino acids in the binding surface towards Fc. Sloan and Hellinga have also described this (Sloan and Hellinga, 1999
). In that particular study, they report a 50-fold increase in Kd for the N34A mutation.
The potential of the single asparagine mutants to function as immobilized ligands during standard industrial procedures was investigated. As revealed from that analysis, Asn36 seems to be the residue most involved in the alkaline sensitivity, since the C2N36A column exhibits a significant increase in stability towards NaOH (Figure 3). An attempt to replace all asparagines in one single mutant was made. Interestingly, this variant was not possible to express in E.coli neither as a single domain nor as a genetic fusion to ABD, possibly due to structural instability. Since C2N36A was shown to increase the alkaline stability remarkably, the substitution of that amino acid was most important. However, since Asn7 easily could be exchanged without negative effects on affinity or stability we chose the double mutant, C2N7,36A, as scaffold for further stabilization. This double mutant showed retained affinity to polyclonal Fc of human IgG (Figure 2A
; Table I
) and also improved behavior in the column analysis (Figure 3
). In addition to the asparagine replacements, we also replaced the single glutamine residue. Glutamines can also be deamidated by the same mechanism as asparagine residues. However, due to the extra -CH2 group, glutamines are not as frequently deamidated as asparagines. This is a result of the strained energy of a six-membered cyclic imide intermediate compared to the five-membered succinimide intermediate of asparagines. Also, the aspartate residues were replaced using C2N7,36A as scaffold. Aspartate can be modified to its isomer but this modification is not observed as frequently as deamidation of asparagine residues due to loss of a good leaving group at high pH (Stephenson and Clarke, 1989
; Tomizawa et al., 1995
). Interestingly, according to the NaOH-exposure experiments, none of the different aspartate or glutamine replacements could further increase the alkaline stability (Figure 3
). Apparently, none of them are modified in this domain.
The structural and stability analyses indicate that the structure is retained for the mutated proteins with only a slight decrease in secondary structure for some variants (Figure 4). Thus, the loss in affinity for C2N34A occurs without major changes in structure. Also, the double mutant shows slightly decreased thermal stability compared to wild-type but retains the ability to refold after denaturation (Figure 5A
). Interestingly, C2N7,36A shows a decreased chemical stability as investigated by the CD analysis (Figure 5B
). Alanine has earlier been suggested to be an amino acid that stabilizes
-helical structures (Horovitz et al., 1992
; Myers et al., 1997
). When substituting Asn34, which is situated in the
-helix, no increased alkaline stability can be detected. Moreover, when substituting Asn36, which is situated in a loop region, for an alanine, an increased stability towards alkaline conditions is detected. It is noteworthy that the structural stability of the selected mutant, C2N7,36A, is lower than for the wild-type protein. Despite this fact, this double mutant reveals a major stabilization towards CIP treatment compared to wild-type C2 (Figure 3
). This indicates that we have specifically stabilized the domain towards alkaline conditions.
IEF reveals that the double mutant gives no deamidated species in contrast to the wild-type C2, which gives a new, more negatively charged variant (Figure 6). Asn34 is involved in the interaction to Fc, and when substituted, the affinity to IgG is remarkably decreased (Figure 2A
). Therefore, this amino acid is not mutated. The single glutamine does not seem to be deamidated when exposed to NaOH. Aspartate residues have been reported to be able to go through an isomerization reaction (Stephenson and Clarke, 1989
; Tomizawa et al., 1995
). However, this modification cannot be detected on an isoelectric focusing gel. However, the HiTrapTM analyses reveal no further stabilization when substituting the aspartate residues (Figure 3
). Accordingly, these residues are not involved in the sensitivity towards alkaline environment of this domain. However, there might be amino acids in the structure that go through an isomerization or deamidation event that do not remarkably affect the activity of the C2 domain.
Finally, genetically fused dimers of C2 and C2N7,36A result in increased binding capacity according to the directed immobilization using the C-terminal cysteine (Figure 2B). Also, the linker region connecting the individual monomers is stable towards NaOH treatment (Figure 3
).
Asn36 is situated in the loop connecting the -helix with the third ß-strand. Numerous studies have reported on the dependence on the sequence and conformation for deamidation (Kossiakoff, 1988
; Lura and Schirch, 1988
; Wearne and Creighton, 1989
). A significant effort has been dedicated to screen the susceptibility of different sequences (Robinson and Robinson, 1991
, 2001
). The most crucial factor is the C-terminal residue succeeding asparagine. The most sensitive sequence is known to be AsnGly. The high deamidation rate for this sequence is due to the small side chain of glycine, which introduces a high flexibility in the backbone of the domain. When a glycine succeeds an asparagine, the main peptide chain and the side chain torsional angles are favorable for the deamidation mechanism. Moreover, these angles occur almost exclusively in the more flexible loop regions (Wright, 1991
; Xie and Schowen, 1999
; Xie et al., 2000
). Asn36 precedes a glycine residue and is also situated in a loop (Figure 1
). Both these parameters are probably affecting the susceptibility towards deamidation. Asn7 is also followed by a glycine residue but does not seem to be deamidated (Figure 1
). A probable explanation for this is that the rigidity of the ß-sheet structure may restrain the flexibility of the individual residues. Asn34 is situated in the
-helix and an aspartate is flanking C-terminal (Figure 1
). Residues in
-helices are less frequently deamidated since the torsional angles are not optimal for the reaction mechanism (Kosky et al., 1999
).
In conclusion, we have stabilized one of the IgG-binding domains of streptococcal protein G towards alkaline treatment. Our protein engineering strategy involves replacement of asparagine residues in order to increase the alkaline stability of a protein. However, each protein needs to be considered on a case-by-case basis since all asparagines may not be possible to replace due to activity losses or structural destabilization. Obviously, some asparagines are not participating in the deactivation process due to structural and conformational parameters. By designing single mutants the susceptible residue can be revealed. With this study we have been able to successfully stabilize the IgG-binding region C2 of protein G and can present a prototype for an improved protein G affinity chromatography medium. Such improvements will be crucial to obtain industrial implementation of protein-based affinity ligands, which normally show too high a susceptibility towards the alkaline conditions used during regeneration of the chromatography devices. This protein engineering strategy might make SPG-based affinity chromatography media useful for cost-efficient industrial-scale purification of monoclonal antibodies for therapeutic use.
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Notes |
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Acknowledgments |
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Received January 28, 2002; revised June 25, 2002; accepted .