Note: Descriptions are shown in the official language in which they were submitted.
WO 93/19091 P~/AVg3/00105
.
- l - 213~321
T~IPARTlTE PUSION PRarBlNS OF GL~THI9NE S-TRANSFE~RAS~3
s
This invention relates to the expression in bacteria such as Esc~le~chia coli
of foreign polypeptides as fusions with glutathione-S-transferase (GST).
In International Patent Application No. PCT/AU88/00164, the disclosure
10 of which is included herein by reference, it is disclosed that a fusion protein
having a foreign po~ypeptide component fused to the enzyrne glutathione-S-
transferase (E.C. 2.5.1.18), preferably to the carboxy-termina~ of the enzyme,
a~oids several of the difficulties associated with known fusion proteins, for
ins~a~ce fusions wherein the foreign polypeptide is expressed as a fusion with
15 E.col~ ,~-galactoidase, in that the GST fusion proteins are generally soluble and
can be purified from bacterial lysates under non-denaturing conditions, for
e~cample by ~finity chromatography on a column of immobilised glutathione. The
QST e~e in the fusion protein may be derived from the parasite helminth
Schistoson~ japonicum, or it may be derived from other species including
20 humans and other mammals.
'rhe GST fusion proteins disclosed in lnternational Patent Application No.
P~T/AU88/00164 rnay be used as such, since the foreign polypeptide component
thereof often retains its antigenicity and functional activity. Alternatively, ~he
25 fusion protein may be cleaved to provide the foreign polypeptide as a synthesis
product, and when the production of such a synthetic polypeptide is desired a
cleavable link may be provided in the fusion protein between the glutathione-S-
transferase component and the foreign polypeptide component. The cleavable
link is preferably one which can be cleaved by a site-specific protease such as
30 thrombin, blood coagulation Factor Xa, or the lilce.
WO 93/19091 PCI/AU93/00I05
2~3~2~
- 2 -
Thus, in one aspect this earlier application discloses a fusion protein
comprising a first amino acid sequence corresponding to the enzyme glutathione-
S-transferase and, preferably fused to the COOH-terminus thereof, a second
amino acid sequence corresponding to a foreign polypept;de (and optionally a
5 cleavable link between these two components), as well as recombinant DNA
molecules, expression vectors, and host cells for use in the production of such a
fusion protein.
In another aspect, this earlier application discloses an expression vector
10 (such as a bacterial plasmid) for use in the production of a foreign polypeptide,
wherein the vector has inserted therein a nucleotide sequence capable of being
expressed as the glutathione-S-transferase enzyrne followed by at least one
restriction endonuclease recognltion site for insertion of a nucleotide sequencecapable of being expressed as a foreign polypeptide fused with the COOH-
15 terminus of the glutathione-S-transferase enzyrne, optionally with a sequencecapable of being expressed as a cleavable link between the enzyme and the
foreign polypeptide.
Preferably, expression of the fusion proteins by the expression vectors is
:: ~
2~ under the control of the tacpromoter which enables inducible, high-level
production ~of these fusion proteins. Preferably a3so, the expression vectors
contain the lac Iq gene, so that they can be used in any E.coli strain.
Polypeptides expressed In E.coli as fusions with GST~ have proven useful
25 for the analysis of prote~n-DNA and protein-protein interactions. Part of thereason for this is that, in contrast to many other expression systems, the
purification of GST fusion~ proteins im~olves non-denaturing eonditions so that the
expressed polypeptide is recovered in a relatively native stale and retains at least
some of its nonn~l properties. Examples include GST fusions with GCN42 and
30 PEA33 that behave at site-specific DNA binding proteins with properties similar
to those of their normal counterparts, while fusions with the retinoblastoma gene
product (Rb)4s, Adenovirus ElA6, c-Kit7 or the TATA binding protein (TBP)8
WO 93/1~091 PCI/AU93/00105
~1~2~21
retain specific interactions with other proteins. In addition, GST fusion proteins
have been used to identify and characterise previously unknown properties of
polypeptides. Thus, nucleotide sequences that are recognised by myogenin
homomers99 MyoDI and c-Mycl~ have been delineated through Polyrnerase Chain
5 Reaction (PCR) amplification of DNA selected by GST-fusion proteins from
mixtures containing random sequences, while fusions with TBP12, Rb4 13 and c-
Myc6 h2ve been used to characterise previously unknown interactions with other
proteins. Such approaches have been extended recently by the use of biotinylatedGST-c-Abl fusions to detect interactions with proteins separated on Western
10 blots'4 and the identification of a protein that interacts with c-Myc through screening of a CDNA llbrary with a radiolabelled GST fusionlS.
In order to simplify the use of GST fusion proteins for such applications, in
work leading to the present invention, the GST expression vector pGEX-2T' has
15 been modified so that it directs the ex~ression of glutathione-S-tran~sferase (GST)
fusion proteins that also contain one or more IgG binding domains from
Staphylococcus aureus protein A. Such tripartite fusion proteins can be detecleddirectly with commercially available antibody-enzyme conjugates, thus sirnplifying
the use of GST fusion proteins as probes for the analysis of protein-protein
20 interactions. In particular, in these fusion proteins the GST COC)H-terminus is
follc~wed by one or more of the five natura]ly occurring IgG binding domains from
Staph~ococcus a~us protein A. Previous studitos have shown that the lgG
binding property of these domains is retained when all five are expressed as a
GST fusion pro~eir.]6 or when they are expressed as isolated domaillsl7 18. It was
25 an object of this modification to provide a non-radioactive ~nd generalised
detection sy~tem using a tripartite fusion protein containing GST followed by one
or more immunoglobulin binding domains and finally the po~ypeptide of in~erest,
where the tripartite fusion protein would also bind to IgG Such a fusion proteincould be purified on glutathione-agarose beads, used as a probe for protein-DNA
30 or protein-protein interactions, and detected using standard antibody-enzyme
conjugates.
. WO 93/1s091 P~/AlJ93/00105
~13232~
- 4 -
The present invention provides a fusion protein comprising a first amino
acid se~uence corresponding to a g}utathione-S-transferase enzyme fused at its
COOH ~erminus with a second amino acid sequence corresponding to a binding
entity, followed by a third amino acid sequence corresponding to a different
S po~ypeptide fused with said binding entity.
In accordance with a preferred embodiment of the present invention3 there
is provided a fusion protein comprising a first amino acid sequence corresponding
to glutathione-S-transferase enzyme fused at its COOH terminus with a second ;
10 arnino acid sequence corresponding to at least one immunoglobulin binding entity
such as an irnmunoglobulin binding protein or one or more binding domains
thereof, followed by a third amino acid sequence corresponding to a different -
polypeptide fused with said immunoglobulin binding entity.
In another aspect, the present invention also provides a recombinant DNA ;molecule comprising a nucleotide sequence which codes on expression for a
fusion protein in which a binding entity is fused with the COOH-terminus of a
glutathione-S-transferase enzyme, followed by a different polypeptide fused withsaid binding entity.
A preferred embodlment of this aspect of the present invention provides a
recombinant DNA molecule comprising a nucleotide sequence which code~ on
expression ~or a fusion protein in which at least one immunoglobulin binding
entity such as an immunoglobulin binding protein or one or more binding
25 domains thereof is fused with the COOH-terminus of a glutathione-S-transferase
enzyme, followed by a different polypeptide fused with said immunoglobulin
binding entity.
If desired, the fusion protein may also include a cleavable link, for example
30 a link which can be cleaved by a site specific protease such as thrombin or blood
coagulation Factor Xa. Such a cleavable link is preferably included between the
&ST moiety and the binding entity; however, a cleavable link may additionally or
WO 93/19091 pcr/Aus3/oolo5
,"~,..................................... 21 3232~ .
alternatively be included between the binding entity and the polypeptide ~used
therewith.
The present invention also provides expression vectors and host cells
S having inserted therein a recombinant DNA molecule in accordance with this
invention, as well as methods of producing the fusion protein of this invention
using such expression vectors and host cells as disclosed in detail in prior
ln~ernational Patent Application No. PCTIAU88100164, the disclosure of which is
incorporated by reference.
`:
In another aspect, the present inv~ntion also provides an expression vector
having inserted therein a nucleotide sequence capable of being expressed as a
glutathione-S-transferase enzyrne followed by a binding entity fused with the
COOH-terminus of said glutathione-S-transferase, and at least one restriction
endonuclease recognition site for insertion of a further nucleotide sequence
capable of being e~pressed as a different polypeptide fused w~th said binding
entiy. Such an expression vector may, if desired, also include a nucleotide
sequence capable of being expressed as a cleavable link as discussed above.
In a preferred embodiment of this aspect, the present invention also
provld~s an expression vector having Inserted therein a nucleotide sequence
capable of being e~pressed as a glutathione-S-transferase enzyrne followed by atleast one immunoglobulin binding entity such as an immunoglobulin binding
protein or one or more binding domains thereof fused with the COOH-terminus
of said glutathione-S-transferase, and at least one restriction endonuclease
recognition site for insertion of a further nucleotide sequence capable of beingexpressed as a different polypeptide fused with said immunoglobulin binding
entity. Optionally, th~s e~cpression vector may also include a nucleotide sequence
capable of being expressed as a cleavable link as discussed above.
W0 93/l9091 ~ 3~ 6 - pcr/Au93/oolos
The GST enzysne in the fusion protein may be derived from Schis~osoma
japonacum, or it may be derived from other species including humans and other
marnmals.
, "
S The binding entity which is included withîn the fusion pro~ein in
accordance with the present invention may be any member of a specific binding
pair, including for example an antigen/specific antibody binding pair (especially
an antigenlspecific monoclonal antibody binding pair), and avidin/biotin bindingpair, or preferably an immunoglobulin binding protein/immunoglobulin binding
10 pair.
~.,
The immunoglobulin binding entity included in preferred fusion proteins in
accordance with this invention may be any protein having immunoglobulin
binding affinity, more particularly binding affinity for immunoglobulin G (IgG).15 Alternatively, one or more immunoglobulin binding domains of such a protein
may be incorporated in the fusion protein. A particularly preferred binding entit~
comprises S~aphylococcus a~lreus protein A or one or more of the lgG binding
domains thereof, however the invention also encompasses the use of other
irnmunoglobulin binding proteins such as Protein Ci, Protein GG and chimaeric
20 Protein A/G. I~ will be appreciated that the immunoglobulin binding protein or
binding domain(s) may~be selected so as to optimise the binding to IgG in the
intended application. Thus~ in view Qf the limited specificity of protein A and in
particular lts poor binding to sheep, goat and rat IgG, other immunoglobulin
binding entlties may be chosen for particular applications. Protein G has a
25 different spectrum of binding (as described by Lew e~.al.~6), and the
immunoglobulin binding entity may even be a synthetic IgG binding domain as
described by Lowenalder e~.al.l9.
The precise nature of the different or "foreign" polypeptide which forms
30 parl of the filsion protein of this invention is not essentiah Accordingly, the
present invention extends to such fusion proteins which incorporate any
polypeptide or protein of interest as the different or "foreign" polypeptide. By
- WO 93/19091 21 3 ~ 3 21 PCr/AU93/00105
way of example, this polypeptide or protein of Interest may be a particular
antigen, with the resuiting fusion protein being useful in a diagnostic test method
and kit for detection of specific antibody to the particular antigen in a sample,
such as a serurn sample, using the well-known enzyme immunoassay (ELA)
S techniques. In such a diagnostic test using a GST/protein A/antigen fusion
protein as the EL~ capture phase, specific antibody if present in the sample will
bind to both the antigenic portion and the protein A portion of the fusion protein
thereby pro~iding a ~larger surface area on the capture phase for binding of theantibody, leading to greater :sensitivity of reaction.
In another example~ of ~ the use of the fusion proteins of this invention in
diagnostlc tests, the polypeptide or protein of interest may be a iireporter'l~entity,
particularly an enzyrne such as alkaline phosphatase, urease, horseradish
pero~dase, or any~other~enzyrne used in colorimetric or chemiluminescent
15 ~ ~determinations. Such a~fusion;protein, for example a GST/Protein A/hors~eradish
pero~dase fusion~protein, can~be used dlrectly in various immunoassay
procedures to detect antigen-antibody reactions by binding to the protein A
moiety, and then dlrectly~detected by standard colorimetric or chemiluminescent
methods. In this way, the requlrement~for various antibody/reporter molecule
20 conjugates (such~as~goat~anti-mouseiHRPO or;rabbit anti-humanlHRPO
con~ugates) for use in~such Immunoassay procedures~can be avoided.
It~will of coùrse~be~appreciated that the inclusion of the GST moiety in the
fusion protein of ;the present~ invention enables the ready purification of the fusion
2~ protein by affinity chromatography on~ immobilised glutathione. In addition,
where the fusion protein is to be used as an ElA capture phase, the fusion
protein may be immobilised on a ~solid substrate by first coating the solid
substrate~with anti-G`ST antibody~and then contacting the coated substrate with
the fusion protein.
Further features of the ~present invention are illustrated, by way of example
~ i:
or~ly, in the following Example and in the accompanying drawings. This Example
WO 93/~9091 PCI/AU93/û0105
21:32321 ~
- 8 -
illustrates the construction of altered pGEX2T expression vectors that direct the
expression of GST fusions containing one, two or three protein A IgG binding
domains. These fusion proteins bind to lgG and can be detected using `
commercially available antibody-enzyrne conjugates. Applications of these vectors
S to the analysis of protein-protein interactions are illustrated by experiments using
an NH2-terminal region of SV40 T antigen as a probe on Western blots of cell
extracts, and by binding assays of immobilised GST fusion proteins probed with
GST-protein A fusions.
"
:
. ~ ,
In the drawings:
Figure 1 shows detection of GST-protein A fusion proteins on
Western blots. Cells transformed with plasmids directing the expression of C;ST- .
prot~in A fusion proteins containing one (a), two (b, pAGEX2T) or three ~c) IgG
binding domains were grown in~the presence of 0.1 mM isopropyl~
thiog~lactopyranoside (lPTG3, and proteins analysed by electrophoresis through
SDS-polyacrylamide gels foll~owed by transfer to nitrocellulose. Polypeptides
containing IgG binding domains were visualised by probing with AP-conjugated
rabbit anti-chicken antibodies, followed by de~ection of enzyrne activity in si~u
~ Figure 2~ shows the structure of pAGEX2T. Indicated in the drawing
~are the IPTG-inducible trp-lac hybrid promoter (Ptac), the regions encoding theSchis~osoma japonicum glutathione-S-transferase (GST), a thrombin cleavage site
and two copies o f the S.aureus protein A lgG binding domain B~PA~
followed by umque BamHl, Smal and EcoRI restriction sites. Also represented
are the position of unique Pstl and EcoRV restriction sites, the gene encoding ,B-
lactamase (ampr), an origin of replication (ori), and an over-expressed allele of
~ the lac repressor (laclq). The nucleotide sequence of a single IgG binding domain
; ~ and the polypeptide that it encodes are also given.
::~: : :
Figure 3 shows the sensitivity of detection of GST-protein A fusion
protelns. Equal quantities of purified GST or GST-protein A ~GST-PA) were
applied to nitrocellulose from left to right as 5 ~11 drops in four-fold dilutions from
WO 93/1sOg1 PCl/AU93/OOtO5
213 2321
g
I ~g to 1 ng. Air dried strlps were ~probed with G53, a monoclonal antibody
directed against GST followed by AP-conjugated~ goat anti-mouse antibodies
(Anti-GST)7 with AP-conJugated~rabbit antlbodies (AP-ConJugated Rabbit Ab),
with either R819, a rabbit antisera raised against Rb (Anti-Rb) or a non-specific
5 rabbit serum (Sigma)~ (Rabbit Serum) followed by AP-conJugated goat anti-rabbit
antib~odies, or with Ab419, ~a~ monoclonal ~ antibody specific for SV40 T antigen
followed by AP-conjugated goat anti-mouse~ antibodies (Anti-TAg). After
washing, enzyme~ acthrity was detected ~ in si~u .
10~ Figure 4 shows the use of GST-protein A-SV40 TAg as a probe for
protein-protein interactions`~on~ Western blots. A Western blot of a HeLa whole
cell extract was probed with ~GST-protein A(a) or GST-protein A-SWO TAg (1-
272) (b) at a concentration of 25 ~g/ml in NT buffer containing 150 mM, 500
mM~or l~ M NaCI. ~After washing~ln the same buffer containing 1~0 mM NaCl,
`~ 15 ~ bound~protein~was~ detecled~ by~ problng with AP-conjugated rabbit antibodies
followed by enzyTne~detection~in si~u .~
Figure 5 shows~ tha~ Rb ls~;amongst the proteins detected by GST- ~ ;
protoin A-SV40 TAg.~ Western~blots~of HeLa~;cells (H) that express Rb and
20 ~ WERI-l cells (W)~that~do not, weré~probed~with a;rabbit antisera raised against
Rb; followed~by AP-coniugated~goat~ ~anti-rabbit antibodies (Anti-Rb) or with GST-
p~ ein A-SV40 TAg (i~27~)~followed~by~a pool~of~antibodies directed against -
SV40 1;~g and ~then ~ conJugated~goat ~antl-mouse antibodies (GST-PA-TAg).
Enzyrne acti~ity~was~ dètéctedi in~ s~
Figure 6 shows the~ bindlng of protein A-TEF-1 to GST-SV40 TAg
;~ immobilisedonbeads.~Purified~GST-Protein~A-TEFl(1-168)wascleavedwith
thrombin and 1,3 or; 10 ~ ncubated~ with glutathlone-agarose beads carrying GST
or GST-TAg (1-260). ~ After~washlng, probe retaiaed on the beads was analysed
30 ~ by probing Western~blots~with~ AP-conJugated rabb~it antibodles and detection of
enzyme activity in situ;. Total represents 1.5 ~ul of ~probe. The triangles indicate
;;the increasing quantity of probe from left to right.
WO 93/19091 P~/AIJ93/00105 ~ ~
2132321
- 10- ,.
Figure 7 shows the binding of complex probes to GST fusion proteins
on beads. Purified GST-protein A~ BP and GST-protein A-SV40 TAg (1-272)
were cleaved with thrombin, mixed together and incubated with glutathione beads .
carr~nng GST or GST-SV40 TAg (1-260) and retained probe analysed as above.
: ~: S Total represents 1/25 of the probe added to each sample of beads.
EXAMPLE .
MAl~RIALS AND METHODS
-; ~; Construction of plasmids.
A single IgG bmdlng~ domain (domain B) of S.aureus protein A20 was
~: isolated by PCR amplification from the plasmid pRlT 5 (Pharmacia2~) using the
oligonucleotldes 5'-TGAGATGTGCGGATAACAAArTCAAC-3' and 5;'-
~CGGATCCl~ GGTGCl-rGAGCATC-3' Amplification was for 20 cycles
of 94C 1 minute, 55C for 1~ minutes and 74C for 13~ minutes using 25 ng
15~ pRIT5 and I00~ng each ollgonucleotide ~n a solution containing 10 mM TrispH: 8.3, 1.5 mM~Mgcl2, 50~ mM KCI, 200 ~m dATP, dCTP, dGTP and
dl~P and~2.5 units T~ermus aquaticus DNA polymerase (Cetus).
The PCR producr was~ lncubated for I hour at 37C with the restriction
2û ~ endonuclease~Sau3A~In~20~ mM Tris-HCl (pH 7.4), 5 mM MgCl2, 50 mM KCl ~and
after electrophoresis~through ~a 2% ~low-gelllng-tempera~ure agarose gel; a 18~ bp
fragment~was:~purified~and:ligated wi~h BamHl cleaved and calf intestinal alkaline
osphatase trèated~pGEX-2T~in: a 10 ~1 reaction containing ioo ng of vector, 50
g~purified fra~ent,~20~mM~ ris-HCI (pH 7.6), ~ mM~:MgCI2, 5 mM
25 dithiothreit~ l`mM::ArFP~;;and l unltT4DNA:ligase~(Pharm~acia). ~After16hoursat 20C the ligation reaction was transformed into competent E.colicells of the
strain H}3101 and :spread onto agar plates containlng 50 yg mM ampiciIlm.
Individual~ colonies àppe~aring after 15 hours growth at 37 C were screened fortheir ability to direct expression of an enlarged glutathione-S-transferase by
30~ inoculat~on into l ml of L-broth.~ growth for 4 hours with agitation at 37 C, and a
further 2 hours growth after the: addition of isopropylthio-B-D-galactoside (IPTG)
~;: to 0.1 mM. ~ Ceils were~pelleled out of a j0 ~11 sample, heated at 90C for 2
~ :`: :
WO 93/19091 2 1 3 2 ~ 21 PCI/AU93/00105
.
- 11 -
minutes in SDS sample buffer and separated by electrophoresis through a 10%
SDS - polyacrylamide gel. Proteins were visualised by staining with Coomassie
Brilliant Blue and a clone identifled that directs the expression of a 33 kDa
polypeptide.
No precipitate was observed in the lane corresponding to cells transformed
with pGEX-2T whlle a faint preclpltate corresponding to the 33 kDa GST
polypeptide indicated that this molecuIe contains a functional protein A IgG
binding domain. By repeating these~cloning steps a plasmid was isolated that ~'
10 expresses a GST fuslon~proteln contalning two proteln A IgG binding domains ~,
which gives much stronger~signal on Western blots when probed :with the antibodyconJugate. Thls plasmld, called pAGEX-2T (see Figure 2), retains the multiple
cloning sites of pGEX-2T in the same frame at the 3' end of the protein A
domains. Repeat~ng~these clomng steps again led to a`plasmid containing~three ,~
15 ~ protein A domains.
Expression of a reglon~of SV40 T antigen encoding amino acids 1-272 as a
GST-protein ~ fusion protein was made possible by PCR amplification of the
plasmid pGEXI-SV40 TAg (1-272)22 with the oligonucleotides S'- ,,
2~ GGATCC~ATGGATAAAGmTAAACAG-3' and
5'-G(~I`(3CATGTGTGAGAGG-3'.~ 1~e PCR ~product was incubated with
BamHl ~and EcoRl~ and;;mserted~ into BamHl and EcoRI cleaved pAGE~2T
producing the~ p}asmid~ GEX2T-SV40 TAg (1-272). A plasmid encoding the
NH2-terminal~168~ammo~acids of T~F-1 as a GST-protein A fusion (pGEX2T- -
25 IEFI (1-168)) was produced ~by inserting a 500.bp BamHI fragment of pGEX2T-
TEF1 (1-426) (described~below) into the BamHl site of pAGEX2T. Similarly, a~
1 kb BamHI-EcoRI fragrnent~of pGEX2T-TBP was Inserted~ mto BamHI an~
EcoRI cleaved pAGEX2T~to generate the ;plasmid~pAGEX2T TBP.
30, A plasmid capable of expressing entlre TEF-~l as a GST fusion protein(pGEX2T-TEF1 (1-426)) was constructed by isolating a 1.3 kb NcoI-BglII
fragment from pXJ40-TEFlA23, treating with the Klenow fragment of E.coli
::~::: : :
WO 93/1gog1 PCr/AUg3/0010s
- 12-
213232~
DNA polymerase I (Klenow) to fill in the ends, followed by litigation with Smal
cleaved pGEX2T. In order to express the first 260 amino acids of SV40 T
antigen as a GST fuslon protein the single PflMI restriction site in pGEX2T was
removed by cleavage and~religation~after incubation with Klenow. A BamHI-
; S EcoRI fra~nent from pAGEX2T-SV40-TAg (~-272) was introduced into this
.
vector, and using the unique PflMl restriction site withln the SV40 coding
sequences, the PflMI-EcoRI fragTnent was replaced;with a PflMI-HindIIl
fragment of pKT26024 to ~produce the plasmid pGEX2T-SV40 TAg (1-260).
Standard procedures were followed~for manipulations of DNA and E.coli
10 transformation. GS:Tfusion proteins were purified from bacterial cultures as
described~
Western I:ransfer and detection Or GST-protein A fusion proteins.
Protein samples~were separated by~ electrophoresis ihrough 10% SDS-
15 ~ polyacrylamide ~gels~ and transferred to nitrooeliulose at 4 C in a buffer containing
25 mM Tris-HCl (pH~8.3), 192~mM glycine and 20% v/v methanol. Transfer was
at 100~ V for 1 hour~or~at 30 V ;for 15 hours, after ~which blots were blocked for 30
minutes~at~20-C in PBS (140 mM NaCl, 3 mM KCl, 8 mM Na2HPO4 and 2 mM
KH2POj)~containing-5%~non-fat dried milk;powder. After~rinsing in PBS, blots
w~ ere probed for 1~ hour ;at 20 C with a 1:5,000 dilutlon ~in ~PBS of an alkaline-
phosphatase :(AP)~ con~ugated~ rabblt anti-chlcken ~ antibody~ (Chemicon). Blotswere~ en~ washed~ three~ times ln~ PBS with enzyme activlty: detected by incubation
in the~ dark~ at 20~(i~ in `a~solutlon containing 100 mM Tris-HCl (pH 9.5), I00 mM
P ~ NaCl, S mM~ MgCl2, 0.33~mg/ml~ nitro blue tetrazolium ~Sigma) and 0.17 mg/ml
25~ ~S-bromo-4-chloro-3-lndalyl~phosphate (Slgma). ~ ~
The sensitivity of ~detectlon of fusion protelns was assessed by do~ blots in
which~ equal quantlties ~of purified GST or GST-protein A were diluted in 150
mM NaCl, 50 mM; Trls-HCI (pH 7.5) (NT buffer) containing 1% bovine serum
30 albumin (fraction V, Sigma) ~(BSA) and applied in four-fold dilutions as 5 1~l
drops to nitrocellulose~strips. After air drying, blots were blocked as aboYe and
probed for 1 hour at 20C with antisera diluted in NT buffer containing 3% BSA.
WO 93/19091 PCr/AU93/00105
2132321
- 13 -
Blots were then rinsed three times in NT buffer, probed for 1 hour at 20C in
NT buffer containing 3% E~SA and a 1:5,000 dilution of AP-conjugated goat anti-
mouse or anti-rabbit antibodies (Promega~, and developed as above.
.
S Probing of Western blots with GST-protein A filsion proteins.
Cell pellets or aliquots of a HeLa whole cell extract25 were heated to 90C
' in sample buffer, separated through 10% SDS-polyacrylamide gels and transferred
~; ~; to nitrocellulose or Immobilon-P (Mlllipore). After blocking as above, blots were
cut into strips and probed~ for 2~hours at 20C with purified GST-protein A or~
10 GST-protein A-SV40 1~4g~ (1-272) at a concentration of 25 )lglml in NT buffercontaining 3% BSA.~ Blots'were washed~three times In NT;buffer and bound
proteins then detected~by~probing with AP-conjugated rabbit antibodies, or with~ a
pool of monoclona! antibodies raised against T antigen followed by AP-
canjugated goat anti-mouse; antlbodles.
Bead binding assays.
Different GST~fusions were purified on glutathione-agarose beads and
stored~at~-20-C~while~st~ bound;~to the beads in PBS contalning 15% glycerol.
Beads~were~ diluted~with ~fresh~ glutathione-agarose beads so as to equalise the20 concentràtion~of;~bound~ pr~atéin between samples. ~ Prior to an assay, 25 ~1 of
be~ were ~ashed in~'a-~mlcrofuge tube with 1 ml of PE~S containing 5~ mM ;~
MgCI2, O.5~Tween 20 and~ 05% BSA, and collected by brief centrifugation~ `
;Thrombin-cleavéd GS~tein~A~fus~on proteins~were added to the drained~
beads~ in 30 ,ul of thé same~ buffer~ and incubated~ for~ 1 hour at 4 C with frequent~
25 ~ agitation.~ Beads~were then;washed twice with I ~ml of buffer without BSA,
drained, and the retained protein~A fusion analy~ed by Western blotting as above.
~ rombln cleavage~ of fusions was ~carried out ~while prnteins were
immoblllsed on beads (200 ~1, containlng up to 200 ~g fusion protein) in 200 ,~
30 buf~er containing 50 mM ~Tris-~CI (pH 8.0), 250 mM NaCl, 50 mM NaCitrate, 1
mM CaC12, 1% BSA and~2 ~g human thrombin~ (Sigrna). After incubation at
20C for 1 hour beads~were~pel} ted and the supernatanl removed. The beads
~ .~
WO 93/19091 2 13 ~ 3 ~1 P~/AU93/00105
- 14-
were washed once with ~Q mM Tris-HCl (pH 8.0) and the combined supernatants
stored at -80C.
".'~
RESULTS
Construction of GST-protein A vector pAGEX2T
A single IgG binding domain of S.aureus protein A (domain B)20 was
isolated by PCR amplification of DNA of the plasmid pRIT521 using ~
oligonucleotlde primers speclfic for domain B and inserted into the BamHI site of -
pGE~2r. The primers were designed such that the BamHI site was re-formed
at a posltion correspondlng to the COOH-terminus of the IgG binding domain so
that further domains could be introduced in the same manner. E.coli
transforrnants were identified containing plasmids that direct the expression ofGST fusion proteins containing one, two or three binding domains. All of these
GST-protein A fusion pr~teins~ were re~ognised when Western blots of cell Iysates
lS were probed with AP-conjugated rabbit antibodies, and the sensitivity of detection
~; ; increased with the number of binding domains (Flgure 1). Correcting for the~ ~ "
different amounts of each ~fuslon pro ein~ the signal lncreases about 50-fold from
; ; one to~two domai~s, and~ about~2-fold~ from two to three domains. Since it was
desired to minimise ~he~size ~of~ the ~GST-protein A carrier and since adding a
20~ ~ird binding domain had a relatively~ minor effect on the sensitivity of detection,
~the plasmid that~encodes two domains ~called pAGEX2T) was chosen for all
tubsequent~work. ~This~vector contains unique BamHl, Smal and EcoRI :-
restriction sites at a~posltlon correspondlng to the COO~-terminus of the secondI gG binding domain and in the same reading frame as in the parent plasmid
pGEX2T, followed~by termination codons in all three frames (Figure 2). Fusions
expressed using this vector consist of GST followed by a thrombin cleavage site,two protem A IgG binding sites and finally the proteln of interest.
Senslti~rity of detection of GST-protein A ~usion proteins.
The sensitivity with which the GST-protein A fusion encoded by
pAGEX2T could be detected was investigated by probing dilutions of purified
protein with different an~isera ~Figure 3). Both GST-protein A and GST alone
WO 93/l90g1 213 ?~ 3 21 Pcr/Au93/oo1os
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were detected by a monoclonal antibody specific for GST (Anti-GST), but GST-
protein A detection was more sensitive, presumably because of the additional
effect of antibody bindlng to the IgG~ blnding domains. A similar sensitivity ofabout 4 ng protein was observed~ for GST-protein A probed with enzy~ne- ~i
5 conjugated rabbit antibodies ~ Conjugaoed Rabbit Ab) and as expected there
was no signal for GST alone. E~nhanced sensitivities of less than 1 ng were
observed when GST-proteln A was probed~flrst with an uncon~ugated antibody
(~abbit Serum, rabbit Anti-Rb or~ MAb419, an Anti-TAg monoclonal antibody)
followed by the appropriate~enzyme-con)ugated second antibody. Amplificat~on
tO :of signal in these càses may~reflect polyvalent binding of enzyme-conjugatedsecondary antibodies~ ta~ primary antibodies bound ~to the protein A domains.
Similar~sensitivities~were~observed for~tripartite fusion proteins containing GST,
.
protein A and portions of SV40 T antigen or of the transcriptional activator TEF-
1. This~suggests~that~ the~presence of ~extraneous polypeptlde~sequences on bothflanks of the protein~A do`mains does not hinder the binding of antibodies.
GSI~p~mA~fusions~as~probes for~Western blots ~ ~
Some appllcatio~ of the pAGEX2T vector can be iliustrated using an
NH2~e~erI~iinal reg~on of~SV40~T antigen (amino~acids 1-272)~that is;known to ;~intera t- ~t sever~ ~dif~nt~protems~ and
~tein~is~ able~to ~autoreg~late- and transactivate the~ SV40;early ~and late
~s~in~v trancipt n~ accions 2~ ADNAfra~entencodl ths ~ :~
region~was~;~inserted ~nto~p~EX2T~resulting~ln~the expression of a tripartite; ;~sion~tein~of 67 ~a.~en this purified fusion~;protein~was used as ~a p e
25~ on Western~blots of a transcript~onally active HeLa whole~ cell extract25 disdnct
bands over a range~ of molecul~ar weights were recognised (Figure 4, 0.15 M, b).Blots incubated with the same probe in solutions contalning ~higher concentrations
of Na~l showed different~patterns~(0.5 M~ 1.0 M) while only faint signals were
obsened on~blots probed~under the same conditions;with~ GST protein A alone
30 ~ (a). ~The strength of~ slgnal; on these blots was reduced with lower concentrations
o f pr~obe while the~pattern obtained was influenced by the type of transfer
membrane, the transfer~conditions, post-transfer renaturation of proteins29 and the
~. ,
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WO ~3/l90gl 2 1 3 2 3 21 P~/AU93/00105 ~
- 16- -~
type of protein blocker and solute~concentrations used during probing (data not
show~
~ .~
ln~o:rder to test the possibillty that the strong signal at about 110 kDa on
S blots probed with SV40 T antlgen in 150 mM NaCi was the Rb protein~ extracts
from HeLa cells that e?~press Rb, and WERI-1 cel]s that have a homozygous
~; ~ deletion of Rb30, were probed with antibodies specific for Rb or with the GST-
protein A-SV40 T antigen~ fuslon~ protein. wheh Immobilon-P was used as the
transfer membrane in such experiments no di~fference in the intensity of the 110 ,
10 ~ kDa~band could be detected between the two cell lines (data not shown).
However, when proteins~ were~ ~transferred to nitroce11ulose, a band was observed
n the~llO kDa region~that~was only present in HeLa cells and that co-migrated
- withla band detected by~anti-Rb antibodies (Figure 5). Nitrocellulose and
; lmmobilon-P have differènt~binding~ properties and it is presumed that a~ species
15 ~present in both cell types Interacts~with the GST-protein A-SV40 T antigen~fusion
~f ~ protein~but~transfers~efficiently only~to lmmobilon-P where it obscures the i nteraction with Rb.
These~expenments~also~reveal several other cellular~proteins that can~
20 ~intera ~ ` th~S 40~T;antlg n ~e e lnteractions are~lndependent of GSTsi~
the~same~species~are~recognised on~blots probed with ~fusion protein that had
beèn~icleaved with throm6in-and consists of the~protein~A :domains fused to the
SV40 T antlgen region,~whiie~lstlnct patterns were observed on blots probed ~
GSI~protein;A fusion protel~ns~containlng port~ons of the transcrlption activators
25~ TEF-I~, ~PEA3 or; TBP (daia ~not~ shown).
Binding of cleaved GST-proteln A fusion proteins ~to GST fllsion proteins on ~ ;
beads. J ` ~ ~
Several studles have used GST-fusion protelns absorbed to glutathione-
30 ~agarose`as an afflnity;matrix~to~purifymolecules from~whole cell Iysa~es467~4 or
from~specifically programmed in vifro translation ~eactions6~2~3. As an extension
to this ~approach, poiypeptides~ expressed~using the~ pAGEX2T vectors have been
~: -
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WO 93/19091 21 3 2 3 2 ~ PCI`/AU93/00105
; - 17 -
used as probes for interactions with GST fusion proteins immobilised on beads.
Such e~eriments require that the GST portion of th~ GST-protein A fusion ;~
protein is first removed by treatment ~,vith thrombin so that the probe does not~; simply absorb to the glutathione-agarose. After incubation of c}eaved probe with
5 ~ beads bearing d~fferent GST fusion proteins, the beads are washed and retained
probe is analysed by Western blotting followed by detection with er~ne-antibody
conjugate.
A probe consisting of the protein A domains fused to the NH2-terminal
10 168 amino acids of TEF-1 does~not bind to beads bearing GST alone but is
retained on beads carrying GST~fused to the first 260 amino acids of SV40 T ;,
antigen (Figure 6). This i;nteraction is specific since a breakdown fragrnent of the
protein A-TEF-1 fusion does ;not~ bind to either type of beads, and the intact
fusion fails to bind to beads~bearing GST fused to NH~-terminal or COOH-
15 tenmnal fragments of TEF-l or to entire TBP (data not shown). Although both
fusions possess DNA bindlng activlties, the interaction does not appear to be
mediated~by DMA since the binding of probe to beads is not affected by the
additlon of DNA to~30~,ug~/ml or of DNAase 1 to 10 ~g/ml. l:
2~ ~ ~Another~illustratlon~of thls~ method takes advantage of cases where two or
moré cieaved GST-protéin~A fusion proteins are of different sizes and so can be
used together in binding ~assays. When cleaved fusion proteins containing protein
A fused to TBP or the ~NH2-terminal 272 amino:~acids of SV40 T antigen were
mlXed and used~ as a probe,~-blnding to~ GST-SV40 TAg (1-260) beads above the
25~ background seen on ~GST~beads~was only observed for protein A-TBP (Figure 7).
;
DISCUSSION
In this Example,~the GST expression vector pGEX2T has been modified in
order to simplify the use of GST fuslon proteins in the analysis of protein-protein
30 interactlons by produclng triparli~e fusion proteins that contain IgG bindingdomains. Polypeptides expressed in this system cnn be purified on glutathione-
agarose under non-denaturing conditions and can be detected without further
.:
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wo 93/lgogl 2 :~ 3~ PCI /AU93/00105
.
- 18 ~
modification using standard reagents and without the use of radioactive materials.
This system is less disruptive and more general than some other methods.
Previous studies have used GST fusions as probes for protein-protein interaction ':,
after labelling by iodinationl53~ or biotinylation~4. Such treatments might . .
~, S sometimes obscure interactions because of the incorporation of labelling moieties ' `,
at binding sites. An alternative non-disruptive method for detecting GST fusion
proteins would be to use monoclonal antibodies specific for GST. Although this ~'~
approach has been used~to probe blots with GST-SV40 TAg ~1-272) and obtain ~,"
results similar to those~obtained with a GST-protein A fusion, the method ~"
10 requires' an addltlonal specific~reagent and~extra manipulations. Such a detection
system~would also not allow the~detectlon of cleaved GST~fusion proteins as ~used "
i n the binding experiments ~described at~ove. ''
Detection of ~GST-protein~A ~fusion proteins require either an enzyme-
linked:antlbody that ltself blnds~to~protein A, or else antibody that binds to '~
protein A~and an approprlate enzyme-linked second antibody. GST-protein A ,~',
fusions have been detected~ directly~with AP-conjugated rabbit antlbodies and
indirectly using mouse~ monoclonal antibodies or rabbit serum; followed by
enzyTne-linked~goat~anti-mouse~or antt-rabbit antibodies.~ Fusion proteins could ~l,,
'20~ also~be detected~ ng~anttbodies from~other species ~such as ~inea-pig, cow,human, ~plg or~ horse, ~ or~ight ~utllise other ~ enzyme-conjugates or include biotin- :
;avidln~ binding~ steps' so ,~as to further amplify the signal. Antlbodies from species
such as the goat, shéep,,rat and chicken bind poorly~ to protein A32 and so could ~,
not be used by themselves.~
The binding, properties~ o f polypeptides may be affected by the presence of
GST and IgG~binding domains in the fusion proteins. Removal of GSTfrom a ,~
tripartite fusion protein containing amino acids 1-272 of S~40 T antigen by
cleavage with thrombin did not ~affect the pattern of bands detected on a Western
30 blot of HeLa~ cell proteins. In~ addition, similar patterns were obtained when the
~ ~ .
same region expressed ~as a GST fusion protein, t)ut lacking the protein A
domains, was used as a probe and detected using a pool of monoclonal antibodies
WO 93~19091 . 2 1 3 2 3 21 PCI`/AU93fOOlO5
;,
- 19 -
directed against GST. GST fusion proteins have been observed to mirror the ;'
properties of their native counterparts in many instances2~I62~3~.
; -~
Previous immunological studles have identified molecules that interact with
5 amino acids 1-272 of SV40 T antigen including Rb26, heat shock protein 70
(hsp70)27 and a protein of 18S kDa28. T he present experiments indicate that E~b
~ .,
is recognised on Western bJots of whole cell Iysates by a GST-protein A fusion ',
.;
protein containing this region of SV40 T antigen (Figure 5). ~Several other
proteins are detected~on these~blo~s, and these may include hsp70 and the l85 ` `~.
10: ~k~a protein as well as~other speci~es ~hat may reveal previously unknown ~ -
Meracdons. The present experiments also suggest that there are direct '~.
interactions between SV40:T antlgen arrd the transcrlption factors TEF-1 and ~ ;
TBP. The possibility that :SV40 T antigen might interact with TEF-1 was
previiously su ested' by experiments` indicating that the abillty of T antigen to ~ .
15; ~ activate ~SV40 transcription is~at::least partially dependent on DNA sequences in ~:'
the:SV40 enhancer to:whlch TEF-1:blnds2233. lnteractions between TBP and
other virus-specified'~transactivators have a!ready been demonstrated for :~
Adenovirus EIA8, Hérpes Simplex virus VP163~ and cytomegalovirus IE2l2. ..
20 ~: .' ;~: Those skilled ln~the~art will appreciate that~ the invention described herein : :~.
is:susceptlbletovarlations~andmodlflcationsother:than:~thosespecifically
des~ribed. It is to be understood that the: invention includes: all such variations :~ - :
and modificatlons :which ~fall ;withln~ its spirit and scope. The~ invention also : .
incIudes:all the steps,:~features:, compositions and compounds referred to or
: 2'5 ~: indicated in ~this ~specification, ~indivldualiy or collectively ?: and any and all
combinatlons of any~two~or more~of said steps or features.
:: ~
WO 93/19091 PCI/AU93/00105
2 1 3 20 ,~
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