From the Department of Biological and Environmental
Science, P. O. Box 35 (YAB), FIN-40014 University of
Jyväskylä, Finland and § Department of
Biochemistry and Pharmacy, Åbo Akademi University, P. O. Box 66, FIN-20521 Turku, Finland
Received for publication, October 19, 2002, and in revised form, November 19, 2002
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ABSTRACT |
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In this study we showed that tetrameric chicken
avidin can be stabilized by introducing intermonomeric disulfide
bridges between its subunits. These covalent bonds had no major effects
on the biotin binding properties of the respective mutants. Moreover, one of the mutants (Avd-ccci) maintained its tetrameric integrity even
in denaturing conditions. The new avidin forms Avd-ci and Avd-ccci,
which have native Chicken avidin and bacterial streptavidin are widely utilized
proteins in many life science applications ranging from purification techniques to modern diagnostics and targeted drug delivery. This methodology, known as (strept)avidin-biotin technology, relies on the
extremely tight and specific affinity (Kd Avidin and streptavidin are exceptionally stable proteins that consist
of homotetrameric antiparallel The avidin (and streptavidin) tetramer is actually a dimer of two
dimers. The monomers that form the tetramer (Fig. 1) interact with each
other in a symmetrical manner and form the three types of
monomer-monomer interactions described in detail by Livnah et
al. (3). The buried surface area between monomers 1-4 (and the
equivalent 2-3) is the largest, whereas the interface area between
monomers 1-3 (and 2-4) is the smallest. The buried surface area
between monomers 1-2 (and 3-4) is also relatively small, but this
interface is important because tryptophan 110 in avidin (Trp-120 in
streptavidin) from subunit 1 participates in biotin binding at the
binding-site of subunit 2, forming the function-related monomer-monomer interface.
A streptavidin mutant with enhanced stability characteristics when
compared with those of the wt streptavidin has been reported previously
(11, 12). The improved stability was achieved by the addition of two
intermonomeric disulfide bridges to the streptavidin tetramer, one
between monomers 1-3 and the other between monomers 2-4, by
changing histidine residue 127 to cysteine. In avidin these interfaces
are similar, and isoleucine residue 117 of avidin is analogous to
histidine residue 127 of streptavidin.
In the present study, we improved the stability of avidin through the
addition of intermonomeric disulfide bridges by the same strategy and
changed isoleucine residue 117 to cysteine. In addition, we produced
two supplementary mutants in which even more intermonomeric disulfide
bridges were introduced. A cysteineless avidin version was also
constructed in which the intramonomeric disulfide bridges of the wt
avidin were removed. The stability characteristics of these mutants
were studied and compared with those of avidin and streptavidin.
Mutagenesis of Avidin cDNA--
Mutations were designed by
using the sequence and structure information obtained from analyses
with the programs GCG (Genetics Computer Group, Madison, Wisconsin),
EMBOSS (European Molecular Biology Open Software Suite), WHAT IF (13),
and InsightII (Molecular Simulations Inc., San Diego, CA). Genetic
engineering of the coding sequence of avidin was performed by
megaprimer (14) and QuikChange (Stratagene) methods using
oligonucleotide primers containing the desired mutations.
Production and Purification of Mutant Avidins--
All of the
mutants were produced by a baculovirus expression system (Bac-To-BacTM,
Invitrogen) in the infected insect cells and purified by
affinity chromatography on 2-iminobiotin-agarose as described
previously in detail by Airenne et al. (15) and Laitinen
et al. (16). The wt avidin was purified from chicken egg
white. Using a Vibra cellTM sonicator, the egg white was
sonicated for 3 min on ice at power setting 8 and 50% duty cycle
with a 1-min break between bursts. After sonication the sample
was diluted with two volumes of PBS and centrifuged for 20 min at
20,000 × g, 4 °C. The soluble fraction was
purified further by affinity chromatography on 2-iminobiotin-agarose as
reported previously (17). Protein samples were concentrated and
subjected to a change of buffers with Centricon YM-3 filters (Millipore, catalog no. 4202). The SDS-PAGE analysis was performed with
a sample buffer without Biotin Binding Assays--
Reversibility of biotin binding was
measured for avidin and the mutants with an IAsys optical biosensor as
reported previously (18). Protein samples were allowed to bind to a
biotin-aminosilane cuvette in PBS containing 1 M NaCl.
After equilibrium was reached, biotin-containing buffer was added, and
protein dissociation was monitored. The affinity of the proteins toward
2-iminobiotin was also determined with an IAsys optical biosensor as
described earlier (9). The biotin binding activity of the mutants
Avd-ci and Avd-ccci after heat treatment was studied with a microtiter
plate assay. Protein samples, at a concentration of 5 µg/ml in PBS, were heated for various time periods at 99.9 °C and then chilled on
ice. The samples were transferred to a Nunc Maxisorp plate and
incubated at 37 °C for 2 h. The wells were washed three times with PBS-Tween (0.05% v/v). The wells were then blocked with PBS containing 1% bovine serum albumin at 37 °C for 30 min and
washed again with PBS-Tween. Biotinylated alkaline phosphatase (Sigma) in PBS, 1% bovine serum albumin was added and incubated at 37 °C
for 1 h. Once again, the wells were washed with PBS-Tween and the
p-nitrophenyl phosphate substrate in DEA buffer (1 mg/ml) was applied to the wells. Absorbance at 405 nm was measured
after incubation for 45 min.
Differential Scanning Calorimetry--
To study the
thermostability of the avidins, a Nano II differential scanning
calorimeter (Calorimetric Science Corporation, Provo, UT) was used. The
protein sample concentration was 0.032 mM (given as the
monomer concentration), and the biotin-containing samples had a
biotin:avidin (monomer) molar ratio of 3:1. The reference cell was
filled with the same buffer in which the sample proteins were dissolved
(100 mM sodium phosphate buffer pH 7.4). The thermograms
were recorded as a function of temperature, between 25 and 130 °C,
at a temperature scan rate of 55.6 °C/h. The base lines were
subtracted, and the Tm values of the
samples were calculated using proprietary software provided by the
manufacturer of the instrument.
Design of the Mutants--
The avidin mutant Avd-nc (C4A, C83Y)
was constructed to obtain information about the importance of the
intrinsic disulfide bridges to the overall stability of the avidin
tetramer. According to the sequence alignment with streptavidin (3),
the cysteine residues were substituted with the same residues that
streptavidin bears in the analogous positions in its primary structure
(Fig. 1, Table I). The avidin
mutant Avd-cci (D86C, I106C) also adopted the cysteines using
the evolutionary approach (18). This
sequence information came from the avidin-like domain of the sea urchin fibropellins (19-22). In the case of Avd-cci, two intermonomeric disulfide bridges, designed to form between monomers 1-4 and 2-3, were introduced, thereby constituting a total of four new disulfide bridges per tetramer (Figs. 1 and
2A). In Avd-cci, cysteine 86 from subunit 1 was presumed to pair with cysteine 106 from the adjacent
subunit 4 and vice versa. Identical contacts were assumed to be present
on the interface of subunits 2 and 3 as well.
Avd-ci (I117C) has an extra cysteine residue in each subunit of the
tetramer. It was designed according to the mutational strategy that
Stayton and co workers (11) and Sano and co workers (12) used to
stabilize the streptavidin tetramer. In Avd-ci, cysteine 117 from
subunit 1 faces cysteine 117 from the neighboring subunit 3. Identically, the corresponding subunit interface 2-4 of the wt protein
contains two equivalent isoleucine residues, which were replaced by
cysteines. Therefore, two intermonomeric disulfide bridges in the
avidin tetramer were expected to form between subunits 1-3 and 2-4,
intensifying the firm association between dimers 1-4 and 2-3 by the
addition of two covalent bonds (3, 4). Finally, to introduce six
intermonomeric disulfide bridges into the avidin tetramer, Avd-ccci, a
combination of mutants Avd-ci and Avd-cci, was constructed.
Purification and Characterization of the Proteins--
The mutant
avidins were produced by a baculovirus expression system (Bac-To-BacTM)
in infected insect cells and purified by affinity chromatography on a
2-iminobiotin-agarose column with a single-step protocol. All of the
mutants showed excellent purification efficiency, indicating that the
mutations had no major effects on the 2-iminobiotin-binding properties.
Moreover, the mutants showed irreversible biotin binding properties
indistinguishable from that of the wt avidin as measured with an IAsys
optical biosensor (data not shown). Affinities toward 2-iminobiotin
were determined for the wt avidin and the four mutants (Table I). The
results indicated that the mutations had not significantly altered the 2-iminobiotin binding characteristics of the mutants. The formation of
the intermonomeric disulfide bridges was studied using SDS-PAGE analysis (Fig. 2B), which confirmed that the cysteines
formed pairs in the manner expected. The biotin binding activity after heat treatment of Avd-ci and Avd-ccci was studied using a microtiter plate assay (Fig. 3). The mutants proved
to be more stable and they remained active, in the sense of binding
biotin, longer than the wt avidin.
Differential Scanning Calorimetry--
To study the thermal
stability of the purified avidin mutants, we subjected them to analysis
by differential scanning calorimetry. The results are shown in a base
line-subtracted form (Fig. 4) and
numerically (Table II). As shown by the
thermograms, Avd-ci was the most stable protein, because its
denaturation Tm value was the highest both in
the absence and presence of biotin. Avd-ccci was only slightly
less stable than Avd-ci. The other mutants and the wt avidin were
equally stable when bound to biotin, whereas Avd-nc and Avd-cci showed
the lowest Tm values in the absence of biotin.
The heat-induced unfolding of the avidins was an irreversible process,
and the unfolding of the proteins was often followed by a more or less
sharp decrease in the heat capacity, most probably because of
aggregation (data not shown).
Avidin and streptavidin are valuable and widely used tools in the
life sciences. In addition to their high biotin binding affinity, the
robustness and flexibility of the system relies on their extreme
stability under various demanding conditions (7). In this study, our
aim was to investigate whether it is possible to increase the stability
of chicken avidin even further without losing its strong biotin-binding
ability. To achieve this goal, two, four, or six intermonomeric
disulfide bridges were introduced to avidin. In addition, we wanted to
clarify the role of the intramonomeric disulfide bridges in the
stability of the avidin tetramer by removing them using site-directed mutagenesis.
According to the DSC results, both the intramonomeric and
intermonomeric disulfide bridges affected the heat-induced denaturation of avidin. Removal of the intramonomeric disulfide bridges from avidin
in the mutant Avd-nc caused a decrease in its Tm
in the absence of biotin ( The most stable of the proteins was Avd-ci, which has an intermonomeric
disulfide bridge in monomer interfaces 1-3 and 2-4. In contrast, the
disulfide bridges created in interfaces 1-4 and 2-3 caused a decrease
in the Tm of the mutant Avd-cci when compared with the wt avidin or even with the cysteineless mutant Avd-nc. This
effect was more prominent in the absence of biotin. These interface
mutations are interesting in that the sea urchin fibropellins (21, 22) were used as a template in their design. We called this kind
of design an "evolutionary approach." These cysteine residue pairs
can stabilize the natural fibropellins, although the arrangement failed
to further stabilize avidin. The reason behind this phenomenon may
return to the fact that the primary structures of avidin and
fibropellins differ significantly from each other. Consequently, they
may have different three-dimensional structures, and the cysteine
residues introduced into avidin according to fibropellins may
not be in the ideal position for formation of a disulfide bridge. The
thermal stability of Avd-ccci (six intermolecular disulfide bridges)
was interesting, because it was lower than that found in Avd-ci (two
intermolecular disulfide bridges) but higher, however, than that of
Avd-cci (four intermolecular disulfide bridges) and of the wt avidin
(no intermolecular bridges). An explanation for the behavior of
Avd-ccci being the sum of its components, Avd-ci and Avd-cci, could be
that the mutation behind Avd-cci causes a small change in the
quaternary structure, and therefore the cysteines originating from
Avd-ci form disulfide bridges with non-optimal bond angles or cause
more steric hindrance to the main chain. The barrel itself has to be
stable, and if, as may well be in the case of Avd-cci, the disulfide
bridges cause some unwanted twisting to the loops, the stability of the
barrel might therefore be jeopardized.
In the presence of denaturing agents it may be preferable to use the
mutants Avd-ci, Avd-cci, or Avd-ccci instead of the wt avidin. This may
be the case particularly for Avd-ccci, because all of its monomers are
covalently linked to each other (directly or indirectly). The mutant is
capable of remaining as a tetramer even after unfolding, as seen in the
SDS-PAGE analysis (Fig. 2B). Therefore it could be
beneficial in applications where a leakage of subunits from
matrix-coupled avidin tetramers would impede the qualitative and
quantitative analysis of molecules. According to the DSC and microtiter
plate assays, the avidin mutants Avd-ci and Avd-ccci, which had
remarkably high Tm values even in the absence of
biotin, could be utilized in PCR protocols, because they can withstand
the temperatures used to denature double-stranded DNA. For example, the
extraction of 2-iminobiotinylated or biotinylated single-stranded DNA
molecules at 95 °C should be possible with Avd-ci- and
Avd-ccci-coated particles.
In most applications of (strept)avidin-biotin technology, high-affinity
biotin binding is essential. Consequently, all of the mutants
introduced in this report are promising candidates for use with
the technology, as they all showed irreversible biotin binding
and high affinity toward 2-iminobiotin. This result was not surprising,
because none of the mutations involved residues that were directly
responsible for biotin binding. However, cysteine 106, which
substituted isoleucine 106 in mutants Avd-cci and Avd-ccci, is located
in the same loop (between We have discovered that the streptavidin tetramer is unstable in
certain conditions without its bound
ligand.2 This could be
detrimental in many applications because of the loss of signal along
with the analytical molecules. Furthermore, it would presumably shorten
the life span of materials if they were to contain covalently linked
streptavidin molecules. Chilkoti et al. (11) as well as
Reznik and et al. (12) have described a streptavidin mutant,
H127C. This mutant has an intermonomeric disulfide bridge
between subunits 1 and 3 (2 and 4), which is analogous to the
isoleucine 117 to cysteine substitution in our avidin mutant Avd-ci.
They report that the resultant mutant is more stable than wild-type
streptavidin (12). Avidin, however, is more stable than streptavidin,
as judged by DSC analysis (6). Therefore, our prediction is that the
1-3 (and 2-4) intermonomeric disulfide bridges bearing mutant Avd-ci
could also have a higher Tm than the
corresponding streptavidin mutant.
The production of streptavidin (or its mutants) is usually performed as
inclusion bodies in Escherichia coli. Its downstream processing includes laborious and time-consuming denaturation and
renaturation procedures followed by the contrived formation of
disulfide bridges (as in the case of the H127C mutant) and additional
purification steps (11, 12, 23-25). In contrast, the production of
avidin mutants, even those with intermonomeric disulfide bridges,
yielded soluble proteins in insect cells that were easily purified in a
single step by affinity chromatography on a 2-iminobiotin column.
Moreover, there are reports describing the production of recombinant
avidin in transgenic corn (26, 27). Interestingly, recombinant avidin
made in corn is less expensive than avidin purified from chicken egg
white (Sigma).
It is possible that avidins with even higher stability than that of
Avd-ci or Avd-ccci could be created. More disulfide bridges could
probably be introduced to link the interfaces, and the amino and
carboxyl termini could be joined together. However, the disulfide bridge strategy used in this study is by no means the only way to
stabilize proteins. Avidin could be stabilized further by using other
strategies such as shortening, to some extent, the loops connecting the
In conclusion, the intramonomeric disulfide bridges of the wt avidin
seem to be an important factor in making avidin so thermostable. On the
other hand, it is possible to enhance the high thermostability of
avidin even further by introducing intermonomeric disulfide bridges.
The most thermostable avidin mutants described in this study therefore
provide more stable tools for avidin-biotin technology, suitable also
for new kinds of applications.
denatured transition midpoints
(Tm) of 98.6 and 94.7 °C, respectively, in
the absence of biotin, will find use in applications where extreme
stability or minimal leakage of subunits is required. Furthermore, we
showed that the intramonomeric disulfide bridges found in the wild-type
avidin affect its stability. The mutant Avd-nc, in which this
bridge was removed, had a lower Tm in the
absence of biotin than the wild-type avidin but showed comparable
stability in the presence of biotin.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
10
15 M) between (strept)avidin and biotin (1,
2).
-barrels (3-5), which upon biotin
binding become even more stable. Transition midpoints of heat
denaturation
(Tm),1
analyzed by differential scanning calorimetry (DSC), have shown that
avidin is more stable than streptavidin in both the absence and
presence of biotin (6). A possible reason for this finding may be the
intramonomeric disulfide bridge found in each avidin monomer (3, 4, 7).
Wild-type (wt) avidin has a high isoelectric point (pI
10.5) and is glycosylated, properties that may cause unspecific
binding in some applications (7, 8). It has been shown that these
unwanted properties can be abolished without markedly affecting the
tight biotin binding affinity or the stability characteristics of
avidin (9, 10).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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-mercaptoethanol.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Fig. 1.
Ribbon representation of avidin tetramer
(stereo view) showing the locations of mutated residues
(sticks). A, subunit color
coding is as follows: green = 1, pink = 2, light blue = 3, black = 4 (numbering
according to Livnah (3)). When the side chains of isoleucine 117 in
each subunit (indicated in yellow) are substituted with
cysteines they form the 1-3 (and 2-4) disulfide bridges in mutants
Avd-ci and Avd-ccci. The blue side chain represents
isoleucine 106 in each subunit, whereas red highlights
aspartate 86; in mutants Avd-cci and Avd-ccci these are substituted
with cysteines to form the 1-4 (and 2-3) intermonomeric disulfide
bridges. White sticks represent the cysteines that form the
intrasubunit disulfide bridges in the wt avidin. In Avd-nc they were
substituted with alanine (Cys-4) and tyrosine (Cys-83). B, a
close-up view from the bottom left corner of panel
A with the same color coding of the subunits.
Description of the proteins used in this study
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Fig. 2.
Formation of the intermonomeric disulfide
bridges. A, schematic representation of the mutants
Avd-ci, Avd-cci, and Avd-ccci displaying the intermonomeric disulfide
bridges. The subunits are numbered according to Livnah et
al. (3). B, nonreducing SDS-PAGE analysis of avidin
(Avd) and avidin mutants Avd-ci, Avd-cci, and Avd-ccci.
Samples were boiled for 15 min in SDS-PAGE sample buffer (without
-mercaptoethanol), and the gel was stained with Coomassie Brilliant
Blue. The wt avidin is found mainly as a monomeric form (the dimeric
form is an artifact seen in several avidin preparations (28)). The
mutants that have intermonomeric disulfide bridges between two subunit
pairs (Avd-ci, Avd-cci) and the mutant that forms a continuous
macromolecule (Avd-ccci) formed dimeric and tetrameric structures in
the manner expected. The double bands in the mutant samples result from
different glycosylation patterns of the recombinant avidins (15). The
lower molecular weight bands are mainly nonglycosylated forms,
whereas the subunits in the higher molecular weight bands bear
sugar moieties. LMW, low molecular weight markers.
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Fig. 3.
Binding activity after heat treatment.
The binding of biotinylated alkaline phosphatase by wt avidin and the
mutants Avd-ci and Avd-ccci after heat treatment (99.9 °C) for
various periods of time was determined by a microtiter plate
assay.
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Fig. 4.
Differential scanning calorimetry.
Heat-induced unfolding of avidin and the mutants in the absence
(A) and presence (B) of biotin in a base
line-subtracted form.
Thermostability analyses
Tm
indicates the difference in Tm compared with that
of the wt avidin in the presence or absence of biotin.
DISCUSSION
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Tm =
8.9 °C),
whereas in the presence of biotin the Tm was
virtually the same as that of the wt avidin. This finding underlines
the importance of the biotin-induced increase in the stability of
avidin. Interestingly, the cysteineless mutant Avd-nc had a slightly
higher Tm than streptavidin
(Tm = 75.5 °C) (6) as an apo form and
significantly higher as a complex with biotin
(Tm = 112.2 °C) (6).
-strands 7 and 8) as the important
biotin-binding residue tryptophan 110. The disulfide bridge formed
between Cys-106 of subunit 1 and Cys-86 of subunit 4 may directly or
indirectly have an influence on Trp-110 and thereby also affect the
biotin binding ability of these mutants.
-strands and by conversion of the glycines in the loops to prolines.
In addition, the introduction of new salt bridges in the interface
areas would also be interesting from point of view of stability.
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ACKNOWLEDGEMENTS |
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We thank Irene Helkala, Pirjo Käpylä, and Jarno Hörhä for excellent technical assistance.
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FOOTNOTES |
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* This study was supported by grants from the Finnish Cultural Foundation.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 should be addressed. Tel.: 358-14-2602272; Fax: 358-14-2602221; E-mail: kulomaa@csc.fi.
Published, JBC Papers in Press, November 20, 2002, DOI 10.1074/jbc.M210721200
2 H. R. Nordlund, O. H. Laitinen, S. T. H. Uotila, K. J. Airenne, E. J. Porkka, N. Kalkkinen, and M. S. Kulomaa, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: Tm, transition midpoint; DSC, differential scanning calorimeter; wt, wild type; PBS, phosphate-buffered saline.
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REFERENCES |
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