Note: Descriptions are shown in the official language in which they were submitted.
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EPITOPE TAGGING SYSTEM
Technical Field
This invention is concerned with an epitope
tagging system and is particularly concerned with a peptide
suitable for use in epitope tagging, an epitope tagged
molecule, a hybrid polypeptide containing the epitope tag,
an expression vector coding for the hybrid polypeptide,
and a process of purifying a protein of interest. The
invention also extends to antibodies specific for the
peptide tag of the invention.
Backqround of the Invention
Epitope tagging, also known-as immunotagging,
epitope flagging, or peptide tagging, is the process of
linking a set of amino acid residues that are recognised as
an antigenic determ;n~nt with a protein of interest (U.S.
Patent No. 4782137; Kolodzie and Young, 1991; Field et al.,
1988; Pati, 1992; Soldati and Perriard, 1991; Geli et al.,
1988; Yee et al., 1987; and Lindsley and Wang, 1993).
Tagging a protein with an epitope allows surveillance of
the protein by a specific antibody, preferably a monoclonal
antibody. This approach can elucidate the size of a tagged
protein as well as its abundance, cellular location,
posttranslational modification and interactions with other
proteins, etc. In particular, epitope tagging allows the
protein to be purified even when there is no method of
assaying its function.
The epitope tagging approach offers significant
advantages over the use of antibodies generated directly
against the protein of interest in that:
13 it saves the time and resources which would
be expended in making monoclonal antibodies
(mAbs);
2) the tagged protein can be monitored with a
well-characterised mAb whose spectrum of
cross-reactivity with non-tagged proteins
is already known;
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3) a comparable negative control can be used,
which is not possible with independently
raised antibodies;
4) the location of epitope in the molecule of
interest can be precisely controlled in
epitope tagging, which can be very
important for structure-function studies;
and
5) epitope tagging may be particularly useful
for discriminating between similar gene
products.
In summary, the epitope tagging approach offers
advantages of universality, precision and economy over the
use of antibodies raised directly against a protein of
interest.
The major uncertainties in an epitope-tagging
strategy are:
1) the ability of the tagged protein to
maintain its biological function, and
2) the influence of target protein sequence on
the antigenicity of the epitope.
For these reasons, it is sometimes essential to
develop epitope tags of short length and different se~uence
characteristics (e.g., different net charges,
hydrophobicity and side groups) to increase the chance of
success in tagging applications.
To date there are only four tagging systems
commercially available, although a number of others have
also been described. Two of the systems contain an epitope
tag of more than 10 amino acid residues. The only
commercial system with an epitope of less than 10 amino
acids residues is the FLAG~ system developed by Hopp et
al. (1988; Prickett et al., 1989). Although FLAG is a
small tag (8 amino acid residues), its highly charged
nature may prevent its application in tagging protein
molecules which are sensitive to net changes in charge.
-
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Table 1 summaries the epitope tag sequences which
have been described in the literature, and compares them to
the pre~erred sequence QYPALT of the present invention.
~, .
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s- ~ , ~,
aO
,_ - a ~o
s - ~ JJ C-- r
~ ~ ~ ~ s~
r- ~ a ~ ~ . . . .
o ~ 1 0
V 2~ H O
o _
~
a) ~ ~ ~ ~ O ,
O
~ ~ ~ ~ W >
o~
- a
~, ~ ~ r~ ~' --' ~ ~ ~ ~
O
11-)
~1
~ ~O
'~ V ~ O~ ~
,C ~, U~ ~ ~ ~ ~ O
~ I U~ O ~D
V O +
t-- V
r ~ 00 r~
L, W
'1E- ~ 5 ~ ~~ r Z ~ 3
w~ ~ ~ s~ ~ a
rci ~v ~ ~ ~ z;
s~ c~r.~ ~ ~ ~
J tV
~ O tn c~
s~ r~ ~ ~ s~ ~ >
' S~ O CJ~
N
v r) 0 ~ a~
/V ~ ,~ r~
c~ (D ~ r-
~ ~ ~ 1 ~ ~ ~' 'C ¢ U ~ ~V
/V ~ ~ (~ ~ V ~ _ 'C r~ -r~ O 'C 'C 'C X
C'~ I I I v ~ , O-~ ~ O-~
W E-~ ~ :> v ~ w V ~ . . . . . . .
SUBSTITUT~ SHEET (RULE 26)
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The present invention seeks to provide an
alternative to the currently available tagging systems.
VP7, a protein of 349 amino acids, is a major
structural protein of bluetongue virus (BTV). Although VP7
is recognised by BTV-specific antibodies in group-specific
serological tests, it cross-reacts with antibodies to
African horse sickness virus and to epizootic haemorraghic
disease virus, and thus strictly speaking is not a
serogroup-specific antigen.
We have found that a hexapeptide having the
sequence Gln-Tyr-Pro-Ala-Leu-Thr (QYPALT), and related
peptides derived from the VP7 molecule of BTV, is
recognised as an antigenic epitope by two mAbs and can be
used as a tag peptide without substantially influencing the
antigenicity of the hexapeptide in the tagged molecule. We
have designated this sequence BTag.
SummarY of the Invention
In a first aspect, the present invention provides
a peptide which functions as an epitope tag when linked to
a molecule of interest, said peptide having the sequence
Q - Y, F, H OR W - P - A, S or V - L or V - T, L, V or Q.
More preferably the peptide has a sequence selected from
the group consisting of QYPALT, QYPSLL, QYPSLQ, QFPALL,
QYPVLV and QYPSLT. Most preferably the peptide has the
sequence QYPALT. Without wishing to be limited by any
proposed mechanism for the observed beneficial effect, we
believe that the amino acids at position 1 and 3 are
critical to the peptide, and that the amino acid at
position 2 should be aromatic.
In a second aspect, the present invention
provides an epitope tagged molecule wherein the epitope tag
is, or includes, a hexapeptide of the invention.
J Preferably the tagged molecule is a protein,
although the invention may encompass any other molecules
capable of being linked to the hexapeptide of the
invention. The protein may for example be an enzyme,
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hormone, genetically engineered single chain antibody
molecules which lack an Fc region, or any other protein.
More pre~erably the protein is a recombinant protein. For
some purposes the epitope may be linked to a carbohydrate.
The tag may be linked to the molecule of interest
by any convenient means. For example, a polypeptide may be
synthesized chemically, using conventional solid-phase
methods, and the peptide tag incorporated into the
synthesis. The tagged polypeptide or protein may be
synthesised as a fusion protein by conventional recombinant
methods, as described herein. The tag may be coupled to a
protein or carbohydrate using conventional cross-linking
agents, such as carbodiimide, or using enzymic methods.
The tagged molecule of the invention allows for
surveillance of the molecule by a specific antibody as
described above. Preferably the antibody is a monoclonal
antibody.
In a third aspect, the invention provides a
hybrid polypeptide comprising a hexapeptide in accordance
with the invention, a protein of interest and one or more
linking sequences of amino acids interposed between said
hexapeptide and said protein of interest , said linking
sequencets) being cleavable at a specific amino acid by a
proteolytic agent.
The proteolytic agent may be an enzyme, such as
enterokinase or a chemical agent such as cyanogen bromide.
A variety of suitable cleaving agents is known in the art.
See for example U.S. Patent No. 4782137; Hopp et al, 1988
and Walker et al, 1994.
The invention, in a fourth aspect, provides a
DNA expression vector comprising DNA coding for a hybrid
polypeptide comprising
1) a hexapeptide of the invention,
2) a polypeptide of interest; and optionally
3) at least one linking sequence of amino
acids interposed between said hexapeptide
and said protein of interest, said linking
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seguence being cleavable at a specific
amino acid by a proteolytic agent.
The invention also provides a DNA expression
vector, comprising DNA coding ~or a hybrid polypeptide
comprising:
1) a hexapeptide according to the invention,
and optionally
2) at least one linking sequence of amino
acids, said linking sequence being
cleavable at a specific amino acid by a
proteolytic agent.
A DNA sequence encoding a desired polypeptide or
protein can be inserted into the DNA of the vector, so that
the hexapeptide and, if desired, the linking se~uence, can
then be co-expressed with a polypeptide of interest. If
the linking seguence is used, the linking sequence is
interposed between the hexapeptide and the protein of
interest, and can readily be cleaved.
The hexapeptide tag in accordance with the
invention may be located at any site in the desired
protein, ie. the N-terminus, at any site within the
sequence, or at the c-terminus of the hybrid polypeptide.
For specific purposes the N-terminus or C-terminus may be
particularly convenient.
In yet a further aspect, the invention provides a
method for producing a hybrid polypeptide in accordance
with the second aspect by transforming host cells with the
DNA expression vector of the third aspect and expressing
said hybrid polypeptide.
The host cell may be anu suitable cell type, such
as a bacterium, a eukaryotic cell such as a yeast, or a
m~mm~lian cell.
In addition, the epitope tag of the invention is
suitable for use in phage display expression systems.
Thus, the epitope tag of the invention may be used to tag a
structural component of a recombinant virus.
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The invention also provides an expression vector
comprising sequences coding for the hexapeptide of the
invention and having one or more cloning sites. The vector
may have multiple cloning site in three reading frames.
In still a further aspect, the present invention
provides a method for purifying or isolating a protein of
interest comprising subjecting the hybrid polypeptide of
the invention to affinity chromatography. The affinity
separation may, for example, be achieved by contacting the
10 hybrid polypeptide with an immobilised antibody, especially
a monoclonal antibody, specific for the hexapeptide tag of
the invention. A variety of coupling methods and solid
supports for the immobilisation of antibodies to form a
support suitable for affinity chromatography are known. A
15 person skilled in the art will be able to select an
appropriate system readily. The purified protein may then
be cleaved from the peptide tag.
In a further aspect, the invention provides
antibodies, including monoclonal antibodies, capable of
20 recognising the hexapeptide tag of the present invention.
Methods for production and screening of polyclonal or
monoclonal antibodies are well known in the art.
Particularly preferred are monoclonal antibodies designated
F10 and D11 as described hereunder. The invention extends
25 to hybridoma cells capable of producing monoclonal
antibodies in accordance with the invention, and to assay
systems, such as competition ELISA assays utilising a
recombinant antigen in which the epitope of the invention
is located adjacent to the immunogenic region of a target
30 protein.
It is envisaged that the monoclonal antibodies of
this invention can be applied not only to purification and J
assay of recombinant or other proteins tagged with the
peptide of the invention, but also to detection of a
35 variety of other types of compounds thus tagged. For
example, environmental monitoring of effluent streams can
be achieved by tagging a component of the effluent or by
SUB~TITUTE S~IEET (RULE 26)
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g
adding a tagged marker to the e~luen~, and assaying
environmental samples for the presence o~ the tag.
The antibodies of the invention may be conjugated
to an enzyme or other signal system commonly used in the
art, for example, horseradish peroxidase, for use in the
detection methods described above.
In another aspect, the invention relates to a kit
comprising an epitope tag in accordance with the invention,
and an antibody to the tag or hexapeptide of the invention.
In a further aspect, the invention provides a kit
comprising a plurality of epitope-tagged molecules, wherein
the tagged molecules are a series of molecular weight
standards or markers. Preferably, the epitope-tag is a
hexapeptide having the se~uence QYPALT. Such a kit would
be useful in Western blot detection of purified proteins to
estimate protein size.
Detailed Description of the Invention
The invention will hereafter be described in
detail with reference to the following non-limiting
examples. In this more detailed description of the
invention reference will be made to the accompanying
drawings in which:
Figure 1 shows deletion mapping in the pGEX
vector. The black arrowed bar on top of the figure
represents the coding region for BTV VP7 with important
restriction sites marked above. abbreviations for
restriction enzymes are: B, Bam~I; Bg, BglII; Bs, BsmI; E,
EcoRI; H, HindIII; N, NdeI; Na, NaeI; R, RsaI; S, Sau3A;
parentheses indicate that the restriction sites were not
present in the original gene se~uence and were generated by
PCT mutagenesis. Three important residues (Cys-15, Cys-65
and Lys-255) are indicated by up-pointing open arrows. The
dotted bars in the centre represent the gene fragments is
given at the bottom in base pairs (bp). Plasmid names are
given at the left, while the corresponding amino acid
numbers (AA#) for each insert in these plasmids are given
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at the right. The reactivity of each recombinant fusion
protein to Dll and F10 is indicated by "+" (reactive) and
(non-reactive).
Figure 2 shows results of SDS-PAGE and Western
blot analysis of GST-fusion proteins. Panel A is a
Coomassie Blue stained gel while panel B is a Western blot.
Lane 1: GST derived from control vector pGEX-lN; Lanes 2-4:
GST-fusion proteins derived from expression plasmids
pGEX-BR, pGEX-BB, and pGEX-BS, respectively (see Figure 1
above).
Figure 3 shows results of ELISA analysis of mAb
binding to overlapping synthetic peptides of various
lengths. The results obtained for Dll are presented in the
left four panels, while those for F10 are shown on the
right. The length of peptides is indicated at the upper
right corner of each panel. The numbers on the Y-axis
represent OD readings, while the residue numbers 1 to 20
given on the X-axis correspond to residues 256 to 275 in
the BTV-l VP7 molecule (Eaton et al., 1991).
Figure 4 provides sequences for nine expression
vectors, indicating the tagged QYPALT epitope and the
multiple cloning sites in three reading frames.
Figure 5 shows results of Western blot analysis
of GST-tag proteins from pGD vectors. Panel A is a
Coomassie Blue stained gel while panels B and C are Western
blots with Dll and F10, respectively. Lane 0: GST
expressed from control vector pGEX-lN; lanes 1-3: GST-tag
proteins derived from pGDl, pGD2 and pGD3, respectively
(see Figure 4 for sequences).
Figure 6 shows a Western blot o~ recombinant VP7
proteins from a previously unknown serotype, designated
BTV-0, and from BTV-15. Panels A and B are Western blots
probed with Dll and F10, respectively. Lane 1: GST-fusion
protein derived from pGEX-BS (see Figure 1 and Figure 2)
used as positive control; lane 2: BTV-0 VP7 expressed from
pET vector (Studier et al., 1990); lane 3: BTV-15 VP7
expressed from the same pET system.
~iUBgTlTUTE StlEET (RULE 2fi)
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Figure 7 shows a comparison of deduced amino acid
se~uences for VP7 molecules of BTV-l, BTV-0 and BTV-15.
Identical amino acid residues are indicated by dots, while
those different from BTV-l are given in one-letter amino
acid codes.
Figure 8 illustrates the mechanism of a
competition ELISA assay.
Panel A shows competition between antibodies
binding to the same epitope.
Panel B shows competition between antibodies
binding to neighbouring epitopes.
Example 1 Identification of Epitope of Bluetonque
Virus VP7 Molecule Recoqnised by two
Monoclonal Antibodies
For structure and functional studies of BTV viral
proteins, a panel of mAbs was generated against the major
core protein VP7.
Mice were ;mmlln;sed with Australian BTV serotype
1 which had been denatured with sodium dodecyl sulphate,
and hybridoma cells were established and maintained as
ascites cells in mice using conventional methods, as
generally described by Lunt et al (1988). Spleen cells
from immlln;zed Balb/c mice were fused with cells of mouse
myeloma cell line Agl4-Sp2/0 (Schulman et al, 1978), using
polyethylene glycol MW1500 (BDH) as fusion agent.
Hybridoma cell lines were screened by indirect
ELISA using BTV antigens which had been partially purified
by centrifugation through 40% sucrose. Subclones were
generated from positive clones by limiting dilution of the
parent lines, and screened by ELISA and Western blotting
using recombinant VP7 produced in yeast in order to
identify positive subclones. Monoclonal antibodies were
purified from mouse ascitic fluid using Protein A af~inity
chromatography.
Among these mAbs, two particular mAbs (Dll and
F10) showed strong binding activity in Western blot
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analysis, indicating that they recognise a linear epitope
on the VP7 molecule.
Using recombinant peptides expressed as fusion
proteins with glutathione-S-transferase (GST) in the pGEX
vectors (Smith and Johnson, 1988), it was shown that both
D11 and F10 ~ind to a 20 amino acid (aa) peptide between
residues 256 and 275 in the VP7 molecule. This is
illustrated in Figures 1 and 2. Further characterisation
of the molecular structure of these mAb-defined epitopes
was carried out using systematic sc~nning of overlapping
synthetic peptides by the PepScan method of Geysen et al
(1984) and by random epitope library methods (Scott et al,
1990; Devlin et al, 1990; Cwirla et al, 1990).
These techniques allowed an epitope consisting of
6 contiguous amino acid residues to be precisely
delineated. In addition, the epitope library made it
possible to identify some of the residues important for
recognition, as well as to predict the presence of a
potentially cross-reactive det~rm;n~nt on EHDV.
For the PepScan analysis, hybridoma cell culture
supernatants of D11 and F10 by ELISA assays were used at
1:20 dilution. Overlapping peptides (5- to 8-mers) were
synthesized and tested for mAb binding. The results,
presented in Figure 3, show that although both antibodies
recognised the penta-peptide QYPAL located at amino acid
positions 259-263, the hexapeptide QYPALT produced higher
signals. mAb F10 also reacted with TAEIFNV, and adjacent
heptapeptide, suggesting that it may recognize a
discontinuous epitope incorporating contact sites on both
peptides, perhaps, juxtaposed by a potential ~-turn (Chou
and Fasman, 1974). PepScan analysis thus showed that both
m~bs recognize the same region of VP7, but indicated that
they may have slightly different specificities. Thus the
main antigenic determin~nt for both mAbs was a 6-aa peptide
with the sequence QYPALT.
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ExamPle 2 Identi~ication of variant seauences
Recoqnised by MAbs
Selection of peptides from a filamentous phage
library displaying random hexapeptides (Scott and Smith,
1990) was used as an adjunct to PepScan for fine epitope
mapping. Affinity purification of binding phages was
essentially as described by Scott and Smith (1990) and by
Parmley and Smith (1988), and was carried out by subjecting
the library to three rounds of biopanning and one of
micropAnn;ng with each of the MAbs. DNA of the affinity-
selected phages was sequenced using the fUSE sequencing
primer (Parmley and Smith, 1988) and the Taq Track
se~uencing system ~Promega) as described by the
manufacturer.
lS After the three successive rounds of biopanning,
phages produced by 93 separate tetracycline-resistant
transductant bacterial colonies were picked ~or micro-
panning to confirm that their displayed hexapeptides were
individually recognised by the respective antibodies. Only
micropanned colonies that yielded more than 100
transductant colonies per 20 ~1 spot were picked for DNA
sequencing. Comparison of their deduced amino acid
sequences showed that MAb D11 had selected phages
displaying three very similar hexapeptides, as shown in
Table 2.
Table 2
Phage Displaying Hexapeptides Selected From
The Epitope Library By MAbS D11 and F10
MAb D11 MAb F10
QFPALL (14) QFPALL (17)
QYPS~L (6) QYPSLL (1)
QYPV~V (1) QWPAVL (1)
SllBSTITlJTE SHEET (RULE 26)
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The number of phage clones displaying each of the
hexapeptide classes is shown in brackets. Residues similar
or identical to the authentic VP7 sequence (QYPALT) at
positions 259 to 264 are shown in bold
Inspection of the amino acid sequence of VP7 of
BTV revealed that these hexapeptide most closely
approximated the residues QYPAL(T) located at positions
259-264 in 8 of the BTV VP7 sequences so far characterized.
We have found that two isolates differ in this region; BTV
0 has the se~uence QYPSLT and BTV-15 has QYPALA. MAb F10
also selected very similar peptides from the library. None
was exactly identical to QYPALT, perhaps since many
hexapeptides may be missing from such a library simply by
chance (Scott and Smith, 1990). Nevertheless, sequences of
the peptides isolated from the epitope library correlated
well with those recognised by the PepScan method. MAb F10,
however, also bound the heptapeptide TAEINFV in PepScan
(Figure 3), while none of the a~finity-purified phages
expressed comparable sequences. A possible reason for this
apparent discrepancy is that if the library contained only
distantly related hexapeptides, they may have had a low
affinity for the paratopes and were thus eliminated during
biopanning. Furthermore, the way in which PepScan and
phage display peptides are presented is probably not
strictly comparable. Nevertheless, both techniques
identified QYPALT, an epitope that is distinct from the
three antigenic regions on VP7 that have previously been
identified (Li and Yang, 1990; Eaton et al, 1~91).
To confirm that their displayed peptides
represented a BTV epitope, phages expressing each of the
affinity selected sequences were propagated, purified as
described by Scott and Smith (1990), and tested by ELISA
with the two MAbs and a polyclonal antiserum raised against
BTV in a rabbit. The results, presented in Table 3, show
that MAb F10 reacted with all four biopanned peptides, but
D11 did not recognize QWPAVL, a sequence that had been
isolated only by MAb F10.
SIJBSTITUTE SHEET (RULE 26)
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Table 3
ELISA Analysis of Representative Recombinant Phages
ELISA A4so nm wi th
Sequence D11 F10 BTV4 As
QFPALL 0.646 0.866 0.392
QYPSLL 0.395 0.467 0.261
QYPVLV 0.525 0.842 0.229
QWPAVL 0.066 0.733 0.128
11 Phage 0.005 o 000 o 053
ELISA plate wells were coated with phage
preparations adjusted to an A288 of 0.25. Monoclona~
antibodies were added at a concentration of 10 ~g/ml and
the rabbit antiserum at a dilution of 1/100. MAbs were
detected with peroxidase-conjugated rabbit antimouse IgG
(Dakopatts) and rabbit antibodies were peroxidase-
' conjugated protein A (Cappel). Results represent averages
of three determ;n~tions.
The epitope library therefore confirmed that the
fine specificities of the two mAbs were different, an
observation further supported by our finding that neither
D11 nor F10 binds VP7 of BTV-15 while F10, but not D11,
binds VP7 of BTV-0. The rabbit antiserum which was
directed against isolate BTV4 also recognised all the
selected peptides, indicating that the QYPALT region was
antigenic in the purified virus preparation used as
immunogen.
In addition to precise epitope mapping,
comparison of the affinity-purified peptide sequences
allows residues critical to the antibody-antigen
interaction to be identified (Scott, 1992). For the BTV-
specific MAbs F10 and D11, Q and P are probably critical,an aromatic residue in position 2 is obviously important,
while position 5 is preferentially occupied by the related
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L or V. The L (or V in one clone panned by D11) at
position 6 also seems to be important in the phage system
(Table 3), which was interesting since in VP7 this position
is occupied by T. Position 4 may not represent a critical
contact residue for either antibody since it can
accommodate an A, V (both aliphatic, non-polar), or S
(polar, hydroxyl). The phage display system does not,
however, accurately reflect the situation in the viral
protein. For example, both MAbs fail to bind VP7 of BTV-15
which has QYPALA in the relevant region. Despite none of
the panned phages displaying a T at position 6, the change
from T to A in the sixth residue of the authentic epitope
is clearly crucial to recognition. This finding implies
that although the random epitope library can identify some
critical residues in a given epitope, its ability may be
limited, perhaps due to the unpredictable effects of
flanking sequences.
Example 3 Confirmation that the hexapeptide QYPALT is
the PrinciPal determ;n~nt for the bindinq
of both D11 and F10
To verify that the hexapeptide QYPALT is the
principal det~rm;n~nt for the binding of mAbs D11 and F10,
synthetic oligonucleotide primers were made in order to
introduce the 6-aa epitope tag into different expression
vectors and to test its antigenicity in tagged recombinant
proteins. This was achieved using oligonucleotide primers
and polymerase chain reaction (PCR; Saiki et al, 1985).
Small PCR fragments encoding the epitope were inserted into
expression vectors. The three classes of epitope tagging
vectors used in this example, designated pD, pTD and pYD,
were derived from three widely used E. col i and yeast
expression vectors: pGD was derived from pGEX-lN (Smith
and Johnson, 1988), pTD was derived from pET-5b (Studier et
al., 1990) and PYD was derived from pYELC5 (Macreadie et
al., 1989; Macreadie 1990). All of these original vectors
contain a uni~ue BamHI cloning site.
-
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The construction process was same for each of the
three classes. To insert the coding sequence for QYPALT
upstream of multiple cloning sites, three small D~A
~ragments were synthesised. Each of the three fragments
differs by a single base pair at the 3'-end of the QYPALT
coding region, so that each of them will generate a
different reading frame for ~usion of foreign genes.
Insertion of these three small gene cassettes into the
uni~ue BamHI site of each of the three original expression
vectors resulted in the nine new vectors, whose se~uences
are shown in Figure 4.
For each class of the three vectors (e.g., pGD1,
2, 3), fusion can be made using any one of the three
possible reading frames for any particular restriction site
present in the multiple cloning site.
The antigenicity of the tagged GST proteins
expressed from the pGEX-derived vectors, pGD1, pGD2 and
pGD3, was tested by Western blot. The results, presented
in Figure 5, indicated that all three GST-tag proteins
maintained the antigenicity of the tag. However, in the
case of pGD1 the antigenicity seemed to be weakened,
probably due to the presence of a highly positively charged
Lys residue downstream of the epitope sequence (see
Figure 4). This indicates that the antigenicity of a given
epitope tag can be affected by the se~uence environment of
the target protein. The results also demonstrated that the
weakening effect in pGD1 was less severe for F10 than for
D11.
Example 4 Application of the taqqinq system in
monitorinq and purification of recombinant
proteins
Having verified that the antigenicity of the
epitope tag in pGD vectors is retained, various tagged
proteins were expressed in the bacteria Escherichia coli
and Bacillus subtilis and in the yeast Saccharo~yces
cerevisiae, and in baby hamster kidney (BHK) cells.
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We have now expressed 23 different proteins using
the epitope tag of the invention. In all of the
experiments, we were able to generate a functional epitope
tag which could be detected by immunological methods, such
as Western blotting, ELISA, immunoprecipitation, or
immunofluorescent microscopy. No cross-reactive protein
bands have been detected in any of the host systems tested.
Moreover, a sequence homology search of public sequence
data banks, including G~nR~nk, revealed no protein
including the sequence QYPALT.
We have observed no adverse affects on either the
expression or the function of the tagged recombinant
proteins, including complex functions such as protein
secretion, membrane insertion, and virus assembly.
We have been able to utilise the epitope tag of
the invention in a variety of expression systems and
recombinant hosts, including Escherichia coli (using both
plasmid and phage systems), Bacillus subtilis, the yeast
~accharo~yces cerevisiae, and m~mmAlian cell lines such as
BHK cells. We consider that the epitope tag of the
invention is generally applicable, and can also be used in
a system such as baculovirus.
In the tagging experiments which we have
performed, the epitope tag has been placed at the
N-terminus, at the C-terminus, or in the interior of the
sequence of the recombinant protein. Our results indicate
that the epitope tag of the invention is a strong antigenic
det~rmin~nt, which can be utilised adjacent to a variety of
flanking sequence environments without significant loss of
antigenicity.
Example 5 C-t~rm; n~l taqqinq
As shown in Figure 4, BTag was initially
incorporated into three different expression systems (the
pGEX vector, the T7 RNA polymerase based pET vector and
pYELC, a yeast expression vector) in all three reading
frames, thus resulting in nine different fusion vectors.
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In the case of the pGD vectors, the BTag was
placed at the C-terminus of the GST protein in the pGEX
vector, so-called "C-t~rm;n~l taggingn. The antigenicity
of BTag in all three vectors has been tested and confirmed
by Western blotting.
When probed with mAb D11, the antigenicity of
BTag in pGD1 was weaker compared with that in pGD2 and
pGD3, probably due to the presence of the Glu(E) and Lys(K)
residues ;mme~;ately downstream (see Figure 5). However,
when mAb 20F10 was used in a Western blot, the difference
was less of a problem. The decrease in antigenicity of
BTag in pGD1 may be overcome by insertion of the GCG (Ala)
codon between the BTag and the Glu(E) codon to introduce
the small.Ala residue, which was present in the native
sequence of BTV VP7 protein, and to maintain the reading
frame.
In addition to these examples of GST-BTag
expression in pGD vector, we have also expressed a
C-termi n~ 1 tagged scFv antibody molecule in E. coli . In
this experiment, the peptide of the invention was found to
be superior to other tagging systems which had been
previously used for expression of scFv, such as FLAG, in
that the antigenicity was stable and no adverse effects
were observed on the expression of the recombinant scFv
molecules.
The C-t~rm; n~ 1 tagging experiments are summarized
in Table 4.
Table 4
Protein Expression vector Expression host
GST pGD E. coli
GST pGD E . col i
GST pGD E . col i
scFv pPOW E. coli
SUBSTITUTE SHEET (RULE 26)
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pPOW is an E. coli vector ~or expression of single chain
antibodies (Power et al; Gene, 1992 113 95-99).
Example 6 N-t~rm; n~ 1 taqqinq
Two groups of N-term; n~l fusions have been
conducted. The first group was obtained by cloning of
target gene (fragments) into pTD or pYD vectors. In these
cases, BTag was not placed precisely at the N-terminus, but
was located at a position 13 and 6 amino acids respectively
~rom the N-terminus. The second group of N-terminal
tagging proteins was obtained by insertion of small double
stranded DNA fragments generated by PCR of overlapping
primers, which code for the BTag sequence at the very
beginning of the protein coding regions, thus forming a
genuine "N-term; n~l tag". Five fusion proteins were
expressed in the first group (three from pTD vectors and
two from pYD vectors). All of them were viral surface
proteins.
In the second group, the BTag was placed at the
N-terminus of filamentous phage fd coat proteins pIII and
pVIII. Four different fusion proteins have been
constructed, two for pIII fusion and two for pVIII fusion.
In these cases, the BTag was placed immediately after the
signal peptide cleavage site. The functional incorporation
of BTag into phage particles indicates that the BTagged
fusion coat proteins were secreted, processed and assembled
as for normal wild-type protein.
The results are summarised in Table 5.
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Table 5
Protein Expression vector Expression host
Classical swine pTD E. col i
fever virus
(CSFV)-gp55
CSFV-gp55 pTD E. col i
CSFV-gp55 pYD yeast
African swine pTD E. col i
fever virus
(ASFV)-p72
AFSV-p72 pYD yeast
rabbit pTD E. col i
calicivirus
VP-60
1~ fd phage pIII fd-tet E. coli
fd phage pIII fd-tet E. col i
fd phage pVIII fd-tet E. col i
fd phage pVIII fd-tet E. col i
fd-tet is a class of vectors derived from the filamentous
phage fd (Parmley and Smith, 1988).
Example 7 Internal Taqqinq
Internal tagging can also be divided into two
groups. The first involved the expression of recombinant
proteins in pGD vectors, while the second group was
obtained by insertion of the BTag coding fragment.
Three viral structural proteins have been
expressed in pGD vectors, forming GST-BTag-target protein
fusions.
In the second group, three different fusion
proteins~have been expressed. Two of them were expressed
in Bacillus subtilis, with the BTag being inserted into the
pro-peptide region of an extracellular protein. The
function of the fusion protein was fully maintained,
~llBSTlTlJTE SHEET (RULE 26)
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indicating that BTag had no effect on protein secretion and
processing. The recipient protein in the third fusion was
a viral membrane glycoprotein and in this instance, BTag
was inserted into a small domain predicted to be exposed on
the cell surface. The fusion construct was expressed in
recombinant vaccinia virus, and the BTag was detected by
immunofluorescent microscopy on the infected cell surface.
The results are summarised in Table 6.
Table 6
Protein Expression vector Expression host
CSFV-gp55 pGD E. col i
bovine viral pGD E. col i
diarrhoea virus
- gP53
BTV-15 VP7 pGD E . col i
neutral protease pNC3 Bacil l us subtil is
neutral protease pNC3 B. subtil is
neutral protease pNC3 B . subtil is
BTV-1 NS3vaccinia virus BHK cells
RCV - VP60 pGD E. col i
pNC3 is a B . subtil is expression vector comprising the gene
for extracellular neutral protease (Wu et al; Gene, 1991
106 103-107).
Example 8 Effect of Sequence Variations on
AntiqenicitY
As mentioned previously, the antigenicity of the
epitope tag was somewhat weakened in pGD1 (Figure 5), but
this effect was less severe with mAb F10 than D11 (compare
lanes 1 in Figure 5B and 5C respectively). Further
evidence comes from Western blot analysis of BTV VP7
~UB~TITUTE SHEET ~RULE 26)
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molecules from two different serotypes, results of which
are presented in Figure 6. While the single point mutation
in the epitope region of BTV-0 VP7 (changing from QYPALT to
QYPSLT, see Figure 7 for sequence comparison) eliminated
the binding of D11, F10 was still able to bind, albeit with
a decreased ef~iciency.
Example 9 Af~initY Chromatoqraphy Purification of
EPitope-Taqqed Protein
An affinity column was prepared using MAb D11 and
Affi-Gel resin (Bio-Rad) using the procedure used by the
manufacturer, and equilibrated with phosphate-buffered
saline, pH 7.2. The recombinant protein was BTV-1 VP7 from
yeast, wllich carries the QYPALT tag within the sequence.
A crude total cell lysate was prepared by
sonication and centrifugation.
The crude cell lysate was prepared in phosphate
buffered saline, the buffer used to equilibrate the column.
The lysate was applied through the column twice.
The column was eluted with 2x bed volumes of 0.5
formic acid, pH approximately 2.5. The eluate was
collected in 8 fractions and neutralised with NaOH.
The ~ractions were assayed by three different
methods:
1) ~D280;
2) ELISA using mAb D11 and a 1:400 dilution of
the elute;
3) Western blot using mAb D11 and 5~1 eluate.
The results are summarised in Table 7.
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Table 7
Fraction # OD280 ELISA Western
reading Blot
(1:400)
1 0.465 0.037 ND
2 0.615 0.021 ND
3 0.600 0.025
4 0.603 0.110 ++
0.630 0.096 ++
6 0.539 0.083 ++
7 0.242 0.072 +
8 0.119 0.000
Although in this prel;m;n~ry experiment no
~uality control was carried out during he preparation of
the column, and the ratio of immobilised Dll to recombinant
antigen was not optimised, the results indicated that Dll
was able to bind recombinant BTV-VP7 under the conditions
used, and that the recombinant protein could be eluted with
formic acid.
Example 10 ComPetition ELISA Assay Usinq Monoclonal
Antibodies of the Invention
Antibody detection is one of the most convenient
methods for diagnosing or monitoring viral or bacterial
infection in hllm~n~ and ~n;mAls~ With the advance of
recombinant DNA technology, recombinant antigens are
nowadays readily available for such purposes. However,
serum antibodies can cause a problem of high background
when assayed directly. As shown in Figure 8, competition-
ELISA (C-ELISA) is based on the fact that the binding of
mAb to a specific antigen can be blocked or reversed by the
competitive binding of serum antibodies to the same epitope
~IJBgT~TUTE SHEET (RULE 26)
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(Figure 3A) or an epitope in close proximity (Figure 8B).
The main difficulty in devising an effective
C-ELISA is the time-consuming process of producing and
screening for a suitable mAb for such application.
We consider that it should be possible to device
C-ELISA by engineering a recombinant antigen with the small
BTag placed next to an ;mmllnogenic region of any target
protein. In this case, the C-ELISA will mainly utilise the
mechanism outlined in Figure 8B, ie. competition between
the binding of mAb to BTag and the binding of polyclonal
antibodies to nearby epitopes on the target antigen.
Two specific examples have been tried:
a) The first example used the recombinant protein
CSFV-gp55, which has the following configuration:
N--T7 tag::BTag::CSFV-gp55tC-tPrm;n~l 1/3 of molecule)--C
When an ELISA assay was performed using mAb Dll,
approximately 60-70~ inhibition was caused by rabbit
anti-T7 tag antibodies (purchased from Novagen). This was
significant, considering that control rabbit serum gave no
inhibition and that this was a "heterologous" competition
system in which the mAb directed epitope was unrelated to
the target epitope being assayed (in this case the T7 tag
epitope).
b) We have also used another recombinant protein, a
GST-fusion protein for RCV-VP60 with the following
configuration:
N--GST::BTag::RCV-VP60--C
When assayed with mAb Dll in competition with
sheep anti-RCV polyclonal antibodies, an average inhibition
of 75% was observed compared with control sheep serum.
We conclude that these results suggest that the
competition had been achieved, and that due to its
heterologous nature the inhibition may never reach 100% as
in the case of homologous C-ELISA.
One of the unique features of the epitope tagging
system of the present invention is that the tag peptide is
recognised by two individual mAbs, yet still maintains its
_
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- 26 -
high specificity. This is very useful in certain
applications when different binding affinities are
required, e.g. in competition-ELISA analysis where
excessive binding of the mAb may prevent competition for
the epitope by antibodies in test sera. Furthermore,
sequence variations flanking or within the epitope tag may
affect mAb binding. Having two mAbs in the tagging system
increases the chance of success in tagging applications
where the failure of binding by one mAb may be offset by
the success of the other.
ExamPle 11 Affinity Columns
Affinity purification columns were prepared using
each of D11 and F10 monoclonal antibodies coupled to
cyanogen bromide-activated Sepharose resin (Pharmacia)
according to the manufacturers' protocols. The optimal
coupling level for each antibody was determined to be
5 mg/ml of resin.
Table 8 shows E. coli recombinant proteins
containing the epitope tag QYPALT which were generated.
Table 8
ProteinDescription Vector Protein QYPALT
sizeLocation
GST Glutathione S PGD3 26 KD C Terr;nAl
Transferase (to GST)
GST-PBC Primary Billiary pGEX-4T 74 KD Amino Acids
Cirrhosis, (between GST and
Autoantigen - GST PBC sequence)
Fusion Protein
LalLa Autoantigen PQE30 45 KDAmino Acids
344-361
Crude cell lysates were prepared for each of the
above proteins by sonication and centrifugation.
500 ~l of each lysate was incubated with 500 ~l
of D11 coupled resin equilibrated with 100 mm phosphate
buffer, pH 7Ø In addition, for GST 500 ~l of lysate was
,
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incubated with 500 ~l of F10 coupled affinity resin.
Lysates and resins were incubated with gentle agitation for
30 minutes at room temperature to allow binding.
Incubation beyond 30 minutes has been shown to result in
reduced yield due to irreversible binding.
Elution was achieved via use of 100 mM glycine
pH 3.5. pH values greater than 3.5 result in reduction in
protein yield.
Elution fractions were analysed for purity,
immunoreactivity and yield by the following methods:
1. OD280
2. Bradford protein estimation
3. Coomassie stained PAGE.
4. Western blot (using mABs D11 and F10
conjugated to horseradish peroxidase).
The results obtained are shown in Table 9.
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WO 97/07132 PCT/AU96/00516
- 28 -
s~ a
~ ~ u~
~ a) . ~
.~ s ~c
v
,, - .
+ a~ a -n
r+ ~ .¢ S~ ~
-~ :+ Z Z ~ v q
.~ ~+
' ~ ~ ~ O C
G v ~ ~
v
p ~
v.~ '~ ~ Q
-,,~ .~ .~ o v
O S~a~ Z Z ~ ~ ~
') ~SA , a) ~ o
u -~
Z s~
~ .~ o
'¢ ~tn V ~ S~
~-~=L O U ~ V
~ (~ ~
V ~ ~ U) U:~
O V ~
-1 C U V
o a
+ ++ + . ~ ~~
~ '' C ~ >1 1)
cp ~ $ ~ v >
-, rn
, V --~ ~ o
>~ un rc
, ~ .,, u~ ~ o 0 rn rn
O ~ ~ ~ ~ .~ 0 V --~
U ~ A Lll A t ~~ Ei U~
oa) o ~ .
o --~ V '~
Z ~
~r~ v o
4 ~ r~l Q IL r-l ~l
4 ~ n r~ o 0 u. s~ :~
¢ ~,~ -t ~ ~ ~ v
O ~ 0 a) .
V
~D ~rn , ~ d~ rn
u a Ul a
a _ ~ 0 un
- ~ 3 3
~>~ -~ L '~i O
,_(O rr. ~ ,~
r~J_ (L un ~ un
Z E~ rr .~ ~10 t~) E~
rn, C~ rn
r~ 1 , 3 1) >1 ~ ~ V
~ ~ ~ 0 ~ un P~ ~
Z ~ ~ ~
CA 02229~40 1998-02-13
w o 97/~7132 PCT/AU96/00516
- 29 -
Although the invention has been described with
reference to the tagging of characterised proteins, the
current advance of molecular biology and the ease with
~ which small tag se~uences can be inserted into any unknown
random cloned gene se~uences by the technology of
polymerase chain reaction (PCR), the present invention
provides an epitope tagging system which is generally
applicable.
It will be apparent to the person skilled in the
art that while the invention has been described in some
detail for the purposes of clarity and understanding,
various modifications and alterations to the embodiments
and methods described herein ma~ be made without departing
from the scope o~ the inventive concept disclosed in this
specification.
References cited herein are listed on the
following pages, and are incorporated herein by this
reference.
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- 30 -
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