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WORLD INTELLECTUAL PROPERTY ORGANIZATION
International Bureau

PCT

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(51) International Patent Classification 7 :

C12N 15/74, 15/11, 15/62, C12P 21/02

(11) International Publication Number: WO 00/09716

(43) International Publication Date: 24 February 2000 (24.02.00)

(21) Internationa] Application Number: PCT/EP99/06022

(22) International Filing Date: 17 August 1999 (17.08.99)

(30) Priority Data:
98115448.7

17 August 1998 (17.08.98)

(71) Applicant (for all designated States except US): EU-

ROPAISCHES LABORATORIUM FUR MOLEKU-
LARBIOLOGIE (EMBL) [DE/DE]; Meyerhofstrasse I,
D-69 1 1 7 Heidelberg (DE).

(72) Inventors; and

(75) Inventors/Applicants (for US only): SERAPHIN, Bertrand
[FR/DE]; Burgunderweg 6, D-69 168 Wiesloch (DE).
RIGAUT, Guillaume [FR/DE]; Zimmer 32, Im Eichwald
18, D-69126 Heidelberg (DE).

(74) Agents: WEICKMANN, H. et al.; Kopemikusstrasse 9,
D-8I679 MQnchen (DE).

(81) Designated States: AE, AL, AM, AT, AU, AZ, BA, BB, BG,
BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM. EE.
ES. FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP,
KE, KG, KP, KR, KZ, LC. LK. LR, LS, LT, LU, LV, MD,
MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD,
SE, SG, SI, SK, SL, TJ. TM, TR. TT, UA, UG, US, UZ,
VN, YU, ZA, ZW, ARIPO patent (GH, GM, KE, LS, MW,
SD, SL, SZ, UG. ZW), Eurasian patent (AM, AZ, BY, KG,
KZ, MD, RU, TJ, TM), European patent (AT, BE, CH, CY,
DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT,
SE), OAPI patent (BF, BJ, CF, CG, CI, CM, GA, GN, GW,
ML, MR, NE, SN, TD, TG).

Published

With international search report.

Before the expiration of the time limit for amending the
claims and to be republished in the event of the receipt of
amendments.

(54) Tide: METHOD FOR PURIFYING PROTEINS AND/OR BIOMOLECULE OR PROTEIN COMPLEXES
(57) Abstract

The present invention relates to a method for detecting and/or purifying proteins and/or biomolecule or protein complexes as well as
fusion proteins, nucleic acids, vectors and cells suitable for this method.

FOR THE PURPOSES OF INFORMATION ONLY
Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT.

Albania

Spain

Lesotho

Slovenia

Armenia

Finland

Lithuania

Slovakia

Austria

France

Luxembourg

Senegal

Australia

Gabon

Latvia

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United Kingdom

Monaco

Chad

Bosnia and Herzegovina

Georgia

Republic of Moldova

Togo

Barbados

Ghana

Madagascar

Tajikistan

Belgium

Guinea

The former Yugoslav

Turkmenistan

Burkina Paso

Greece

Republic of Macedonia

Turkey

Bulgaria

HI)

Hungary

Mali

Trinidad and Tobago

Benin

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Mongolia

Ukraine

Brazil

Israel

Mauritania

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United States of America

Canada

Italy

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Central African Republic

Japan

Niger

Viet Nam

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Netherlands

Yugoslavia

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C6te d'lvohe

Democratic People's

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China

Republic of Korea

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Czech Republic

Saint Lucia

Russian Federation

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Sri Lanka

Sweden

Estonia

Liberia

Singapore

WO 00/09716

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PCT/EP99/06022

Method for Purifying Proteins and/or Biomolecule or Protein Complexes

Description

The present invention relates to a method for purifying substances such as
biomolecules, proteins, protein and/or biomolecule complexes, subunits of
biomolecule complexes, cell components, cell organelles or even whole
cells. It also concerns fusion proteins for use in this method and other
related subjects.

Protein expression and purification methods are essential for studying the
structure, activities, interactions with other proteins, nucleic acids etc. of
proteins of interest. Methods that are currently available use systems such
as bacteria or cells transfected with expression vectors or infected with
bacculovirus.

In order to study individual proteins a first requirement is to obtain sufficient
amounts of that particular protein to be able to carry out biological and
biochemical analyses such as activity tests, interaction assays, structure
determination and the like. For this purpose the genes coding for the
proteins of interest are cloned into vectors that allow the expression of
those proteins in suitable host cells. Usually the proteins are expressed at
high levels. This over-expression leads to the generation of large amounts
of protein but often has the disadvantage of yielding insoluble protein which
is present in so-called inclusion bodies in the cells. The over-expressed
protein then has to be resolubilized before analysis. Although such methods
work well for conventional protein detection methods based on weight
analysis (polyacrylamide gels, Western blots, etc.) of the expressed protein,
they are not suitable for other studies and for assays on protein complexes.

PCT/EP99/06022

WO 00/09716

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° UI o«-e~- „.v„, es sed in bacteria. Apart trom

f „,.xa mP ia.wh.nau k a^cp,o«e,nsaraaxp,asaad,

processing or glycosylation.

proteasome, Swafie.d et a... 1996, Nature 379, 658).

♦ ♦woir hasal level is therefore

easily ava.lable because of 9 . fication scheme requires

^hii^h a suitable protocol. Developing a punf.cat.on sc
establish a sunaDie h t h P taraet prote n by a

) analyses have to be repeated for each protein.

r: a e ill-P-e. in an „«v ca-n =
leHa, tna. apa*a»v «. *. a,r,ni,y - ^llTIt
prot ein ,o «ha m a,nx via *e a,«V « > — be

WO 00/09716 PCT/EP99/06022

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conditions are needed for the subsequent elution wich can often destroy,
damage or denature the protein of interest.

Affinity tags possess groups or moieties which are capable of binding to a
5 specific binding partner with high affinity. Various affinity tags are known
in the art and have been widely used for the purification of proteins.
Examples are the IgG binding domains of protein A of Staphylococcus
aureus, glutathione-S-transferase (GST), maltose binding protein, cellulose
binding domain, polyarginine, poiycysteine, polyhistidine, polyphenylalanine,

1 0 calmodulin or calmodulin binding domains. These bind with high affinity to
an appropriate matrix which is covered with the specific binding partner. In
the case of protein A, IgG-coated sepharose has been used for affinity
chromatography of fusion proteins possessing a protein A domain (Senger
etal., 1998, EMBO J., Vol.17, 2196-2207). Other examples are discussed

15 in Sassenfeld, TIBTECH, 1990, p. 88. A plasmid vector containing a
cassette encoding a calmodulin binding peptide is available from Stratagene.

Normally, fusion proteins are tagged with only one affinity tag and are
purified in a single purification step. This often leads to problems due to

20 remaining contaminants. Another limitation of most of the conventional
methods is that they are adapted for expression of the proteins in bacteria
only. WO96/40943 discloses a method of expressing fusion proteins in
gram-positive bacteria either anchored to the membrane or in secreted form.
The anchored proteins are cleaved off using TEV protease and subsequently

25 affinity purified via an affinity tag.

Often the affinity tag is removed from the fusion protein after the affinity
purification step by the action of a specific protease such as the TEV
protease. This means however, that the purified fractions contain
30 substantial amounts of this protease (Senger et al. 1998) which severely
limits the applications of such protein preparations.

PCT/EP99/06022

WO 00/09716

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„ is therefore an object of the present invention to provide a
11 —on method for pro.eins and/or biomo,ecu,e or protern
col es =nd/or components or subunits hereof which atimina.es *e
rental of the currentfv known methods and afiows efficent

One method according to the invention for purifying substances s ~~d
from proteins biomolecules. complexes of protarns or bromolacules,
run::"!,. — components. ce,, organe„ee. and whoie cetts

TZZZZZ* — — - - t

one or mora eubuniu o, a biomotecute comptex. the potypephdas or

Staphylococcus protein A,
, bl maintaining the expression environment under condmone that
' ' TaTata expression of the one or more peptides or subumts ,n

a native form es fusion proteins with the affinity tags,
w erecting and/o, purifying the one or more ^^^Z
bY a combination of a, ieas, two different affimty punfrcahon tep
each comprising binding the one or mora poiypeptides or
one J*, teg ,0 a supper, materia, capebie o,
one o, the affinity tags and separating the on. o, more p VP ephd
a, aubunits from the supper, materia, after subs.ances no, bound
the supper, malaria, have been removed.

An a„erna,ive me,hod o, the invention which is par,icu,ar,y suhabie for
30 detecting and,or purifying P-otein o, biomo,ecu,e comp,axes ,s a method
comprising the steps:

WO 00/0971 6 PCT/EP99/06022

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(a) providing an expression environment containing one or more
heterologous nucleic acids encoding at least two subunits of
a biomolecule complex, each being fused to at least one of
different affinity tags, one of which consists of one or more

5 IgG binding domains of Staphylococcus protein A,

(b) maintaining the expression environment under conditions that
facilitate expression of the one or more subunits in a native
form as fusion proteins with the affinity tags, and under
conditions that allow the formation of a complex between the

10 one or more subunits and possibly other components capable

of complexing with the one or more subunits,

(c) detecting and/or purifying the complex by a combination of at
least two different affinity purification steps each comprising
binding the one or more subunits via one affinity tag to a

15 support material capable of selectively binding one of the

affinity tags and separating the complex from the support
material after substances not bound to the support material
have been removed.

20 For the purpose of this invention, a biomolecule can be a protein, peptide
or a nucleic acid or other biomolecule. A biomolecule complex denotes a
complex of at least two biomolecules, preferably at least one protein
associated either with other proteins which are then called subunits or with
other substances which can for example be nucleic acids. The biomolecule

25 complexes can be natural ones such as nuclear snRNPs or antigen-antibody
complexes, or they can be artificial ones such as mutant DNA binding
proteins associated with mutant target DNAs. Any complex molecule
comprising as one or more subunits a polypeptide or subunit expressed
according to the invention and/or further comprising other components

30 which associate in a manner stable enough not to be dissociated by the
affinity steps is a biomolecule complex that can be detected and/or purified

PCT/EP99/06022

WO 00/09716

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bv the method of the invention. A protein complex gene,*, devotes a
complex between protein subunits.

The nucieio acid seguence ot the protein to ba purified must be known or
« east avaiiabie ao that K can ba Coned into a nucieic acid whrch is
uitabie ,o drive expreasion in the appropriate boa, ce»s o, celi-fre
expression aystema. H a protein compfex ,a to ba purified, the nucieic acd
seouence of a. leas, one of its subunits baa to be Known or avertable.

The heterologous nucieio acid driving the expression o, .he protein to be
purified accord,ng,o<he,nven«ion,huscon,a,ns appropriate seguencea that
Low ,. to be maintained in the chosen host caf, or cefi-free system, such
Z promoter and, if necessary, other control aeguences such as enhancers
and poly A sites.

,„ principle any host cel, tha, is compatible with the hetero^us nuc*
aoi d from which .he po.ypep.ides o, subunits are .o be expressed s surta«e
as an expression environment. These ca„s can ba prokaryobc ce« such s
bacteria e.c. or euxaryobc c.l,e auch as yeas, fungi or "-"-"^
Preferabty. the pro.ein or subuni. o, protein complex to be pur f,d
expressed in ,«s nature, host. Since this method ,s very efferent, he
p Ins are preferably expressed a, thai, besai leveis.This has .he
tntaga of avoiding .ha formation of inclusion bodies and aiso reduce
he risx o, .oxic effects on .he ce„ .ha. iarge amoun,s of certain protarn
mav have. Furthermore, this avoids purifying excess protem subun, s the
1 no, assembled into a compiex or ,ha, are asaembied ,n,0 aberrant
complexes (see above).

A,,e, ,he heterologous nucleic acid encoding ,he fusion protein has been
Educed in,o a chosen boa, ce„ ,ha cel, is cultured unda, condrbons
which allow ,he expression of ,he fusion pro,em(sl.

WO 00/0971 6 PCT/EP99/06022

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As already mentioned, the transcriptional control sequences are preferably
selected so that the fusion protein is not over-expressed but is expressed
at basal levels in the cell. This serves to ensure that the protein is expressed
in a native form. Native form means in this context that a correct or
5 relatively close to natural three-dimensional structure of the protein is
achieved, i.e. the protein is folded correctly. More preferably, the protein
will also be processed correctly and show normal post-transcriptional and
post-translational modification. The correct folding is of great importance
especially when the expressed polypeptide is a subunit of a protein complex
10 because it will bind to the other subunits of the complex only when it is
present in its native form. However, it is also possible to express mutant
proteins. These can also have a native conformation. Such mutant subunits
can, for example, be used to purify mutant complexes, i.e. complexes that
contain some other mutated subunits.

Depending on the protein or subunit to be purified, the fusion protein is
expressed intracellular^ or secreted into the culture medium. Alternatively,
it might be targeted to other cell compartments such as the membrane.
Depending on the protein an appropriate method is used to extract the

20 fusion protein from the cells and/or medium. When a fusion protein is
expressed which is targetted to a certain subcellular location, e.g. the
membrane of cell organelles or the cell membrane, these organelles or the
cells themselves can be purified via the binding of these membrane
proteins. It is also possible to purify cells or cell organelles via proteins

25 naturally expressed on their surface which bind to the fusion protein of the
invention.

Further, it is possible to purify biomolecule or protein complexes/subunits
or other substances that are capable of binding to or completing with the
30 fusion protein generated according to the invention. These substances can
bind to fusion protein either directly or via linker mediators. Linker mediators
in this context may be anything which is capable of binding two or more

PCT/EP99/06022

WO 00/09716

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biomolecules so that these biomolecules are then part of a complex
although they may not be directly associated with each other.

According to the invention it is also possible to use cell-free systems for the
expression of the polypeptides or subunits. These must provide all the
components necessary to effect expression of proteins from the nucleic acid
such as transcription factors, enzymes, ribosomes etc. In vitro transcription
and translation systems are commercially available as kits so that it is not
necessary to describe these systems in detail (e.g. rabbit reticulocyte lysate
systems for translation). A cell-free or in vitro system should also allow the
formation of complexes.

For the purification according to the invention it is preferable to employ
affinity chromatography on affinity columns which contain a matrix coated
with the appropriate binding partner for the affinity tag used in that
particular purification step.

In accordance with the method of this invention two affinity steps are
carried out. Basically each affinity step consists of a binding step in which
the previously extracted protein is bound via one of its affinity tags to a
support material which is covered with the appropriate binding partner for
that affinity tag. Then unbound substances are removed and finally the
protein to be purified is recovered from the support material. This can be
done in two ways. The first possibility is to simply use conventional elution
techniques such as varying the pH or the salt or buffer concentrations and
the like depending on the tag used. The second possibility is to release the
protein to be purified from the support material by proteolytically cleaving
off the affinity tag bound to the support. This way, the protein can be
recovered in the form of a truncated fusion protein or, if all affinity tags
have been cleaved off, as the target polypeptide or subunit itself.

WO 00/09716 PCT/EP99/06022

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According to one embodiment of the present invention a fusion protein of
a single polypeptide plus two different affinity tags is expressed, wherein
one of the tags comprises one or more IgG binding domains of protein A of
Staphylococcus aureus.

More preferably, a specific proteolytic cleavage site is present in the fusion
protein between the one or more polypeptides or subunits and the one or
more affinity tags so that proteolytic cleavage allows the removal of at least
one of the affinity tags, especially the IgG binding domains of protein A.

Proteolytic cleavage can be carried out by chemical means or enzymatically.

The proteoloytic cleavage site that is used to cleave off one of the affinity
tags is preferably an enzymatic cleavage site. There are several proteases

15 which are highly specific for short amino acid sequences which they will
cleave. One of these is a specific cleavage site of Tobacco Etch Virus
(TEV), which is cleaved by the TEV protease NIA. Recombinant TEV
protease is available from Gibco BRL. The TEV cleavage site is preferably
used as the cleavage site to remove the protein A domains from the fusion

20 protein.

Even more preferably, the affinity step using protein A binding to IgG is
carried out first by binding the one or more polypeptides or subunits via the
one or more IgG binding domains of Staphylococcus to a support material

25 capable of specifically binding the latter, removing substances not bound
to the support material and separating the one or more polypeptides or
subunits from the support material by cleaving off the IgG binding domains
via the specific proteolytic cleavage site, and then another affinity tag is
used to purify the protein further via a conventional elution step comprising

30 binding the polypeptide or subunit via another affinity tag to a second
support material capable of specifically binding the latter, removing

PCT/EP99/06022

WO 00/09716

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substances not bound to the support material and separating the
polypeptide or subunit from the support material.

When the proteins are present at low concentrations in the expression
environment and on the support material, a .arge amount of protease ,s
required to reiease the bound materia, from the support material. In other
words, when the substrate concentration is low a high level of enzyme ,s
required to drive an efficient proteolytic reaction. The second purificauon
step is then important to remove remaining contaminants and the protease.
Removal of the protease is preferable in order to eliminate any negahve
influences of the proteolytic activity on the preparation.

However, in some cases it may be desirable to remove all the affinity tags
,n which case it is also possible to utilise two or more different proteolytic
cleavage sites for the separation of the polypeptide/subunit of interest from
the support material.

The method according to the invention not only facilitates efficient
purification of proteins of interest but also allows fishing for and detecfng
components present in complexes with which the polypeptides or subumts
are associated or complexed either directly or indirectly, e.g. molecules such
as linker mediators. This would allow selective fishing for certa.n
substances which may be potential drugs even from complex mixtures.

According to a second embodiment of the invention it is possible not only
to detect or purify the subunit containing fusion proteins expressed but also
other substances that are capable of associating with the prote.ns
expressed in a direct way. i.e. by directly binding to the fusion
indirectly via other molecules to form biomo.ecule complexes. If a fus.on
protein of a subunit of a biomolecu.e or protein complex is punf.ed
according to the invention the affinity steps are chosen so that other
complex components which have bound to the fusion protein are st,l.

WO 00/09716 PCT/EP99/06022

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associated with the subunit after the purification steps so that they can be
detected/purified as well.

The biomolecule complexes can be formed in the expression environment
5 such as cellular complexes. Alternatively, other complex components may
be added to the subunits already expressed to form complexes in vitro or
may even be added when the subunit containing fusion protein is already
bound to a support material in an affinity step.

10 It is also possible to express two (or more) subunits of the same complex
each as a fusion protein with a different affinity tag. When the subunits
associate they can be detected/purified possibly together with other
complex components by a series of affinity steps in which each time the
complex is bound via a differently tagged subunit. The two or more affinity

1 5 tags can be fused with a single subunit of a complex or with two or more
subunits which bind to each other or are simply present in the same
complex.

The purification steps can be carried out as described above.

Some polypeptides are present in more than one complex so that the
components of all complexes can be purified.

If one is interested in a single complex A one can also subtract other
25 complexes B that also contain one of the subunits of A by fusing that
subunit of A to one tag and fusing a subunit unique to B with a different
tag. The tagged subunit of B will bind to a specific support material. If the
fraction not bound to that support material is used in the second affinity
purification step, complex B will no longer be present because it was
30 removed (subtracted) in the first affinity step. Many similar scenarios can
be envisaged and designed by a person skilled in the art.

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Further affinity tags in addition to the IgG binding domains that can be used
in accordance with the present invention can be any conventional affinity
tag. Preferably, the second affinity tag consists of at least one calmodulin
binding peptide {CBP). Calmodulin binding peptide as an affinity tag has
been described and is commercially available (Stratagene). When a
calmodulin binding peptide is used the corresponding purification step is
carried out using a support material that is coated with calmodulin. The
calmodulin binding peptide tag binds to calmodulin in the presence of low
concentrations of calcium. It can subsequently be eluted using a chemical
agent such as a chelating agent like EGTA. Preferably, around 2 mM EGTA
is added for the elution step.

Another aspect of the present invention is a fusion protein consisting of one
or more polypeptides or subunits fused to at least two affinity tags, wherein
one of the affinity tags consists of at least one IgG binding domain of
Staphylococcus protein A.

Other fusion proteins according to the invention are those additionally
including a proteolytic cleavage site, preferably to cleave off the IgG binding
domains, or those in which the second tag represents one or more CBPs.
Again, the skilled person will be able to select and construct the most
suitable combinations of tags and cleavage sites and polypeptides and/or
subunits in fusion proteins depending on the affinity strategy used. The
fusion protein can be constructed so that the above-mentioned purification,
detection or fishing procedures can be carried out.

There are several possibilities for constructing the fusion protein. In
principle, the affinity tags may be fused close to either of the N- or C-
terminal ends of the polypeptide(s) or subunit(s) to be expressed. The order
in which the tags are fused with the polypeptide(s) or subunit(s) is not
critical but can be chosen according to the affinity protocol to be used.
Small peptides such as the CBP can even be fused to the polypeptide(s) of

WO 00/09716 PCT/EP99/06022

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interest internally (as long as the reading frame on the nucleic acid is not
changed). Preferably, the tags are located near to the same end of the
polypeptide(s) or subunit(s), wherein it is especially preferred that the IgG
binding domains are placed at the N- or C-terminus of the complete fusion
5 protein, followed by a proteolytic cleavage site, the other tag{s) and the
polypeptide(s) or subunit(s).

The fusion protein can also contain a second proteolytic cleavage site for
the removal of the second affinity tag. The most preferable combination of
10 affinity tags and cleavage sites is the one with protein A domains of
Staphylococcus as the first affinity tag which can be cleaved off via the
TEV protease and using at least one calmodulin binding peptide as the
second affinity tag which allows the elution of the truncated fusion protein
using a chelating agent such as 2 mM EGTA.

Another aspect of the present invention is a heterologous nucleic acid
coding for a fusion protein as the one described above.

A further aspect of the invention is a vector comprising at least one
20 heterologous nucleic acid coding for a fusion protein of the invention. This
vector contains the nucleic acid under the control of sequences which
facilitate the expression of the fusion protein in a particular host cell or cell-
free system. The control sequences comprise sequences such as promotors,
and, if necessary enhancers, poly A sites etc. The promoter and other
25 control sequences are selected so that the fusion protein is preferably
expressed at a basal level so that it is produced in soluble form and not as
insoluble material. Preferably, the fusion protein is also expressed in such
a way as to allow correct folding for the protein to be in a native
conformation. Preferably, one or more selectable markers are also present
30 on the vector for the maintenance in prokaryotic or eukaryotic cells. Basic
cloning vectors are described in Sambrook et al., Molecular Cloning,
Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory

PCT/EP99/06022

WO 00/09716

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Press, ( 1 989) . Examples of vectors are plasmids. bacteriophages, other viral
vectors and the like.

In a preferred embodiment vectors are constructed containing pre-made
cassettes of affinity tag combinations into which the nucleic acid coding for
the polypeptide or subunit of interest can be inserted by means of a multiple
cloning site such a polynucleotide linker. Thus, a vector according to the
invention is also one which does not contain the coding sequences for the
polypeptide(s) or subunit(s) of interest but contains the above-mentioned
components plus one or more polynucleotide linkers with preferably unique
restriction sites in such a way that the insertion of nucleic acid sequences
according to conventional cloning methods into one of the sites in the
polynucleotide linker leads to a vector encoding a fusion protein of the
invention.

In a further preferred embodiment the vector comprises heterologous
nucleic acid sequences in form of two or more cassettes each comprising
at least one of different affinity tags one consisting of one or more IgG
binding domains of Staphylococcus aureus protein A, and at least one
polynucleotide linker for the insertion of further nucleic acids. Such a vector
can be used to express two subunits of a protein complex, each tagged
with a different tag.

Vectors according to the invention can be introduced into host cells stably
or transiently, they can be present extrachromosomally or integrated into
the host genome, and they can be used to produce recombinant cells or
organisms such as transgenic animals.

Another object of the invention is a cell containing a heterologous nucleic
acid or a vector of the invention. These cells can be prokaryotic or
eukaryotic cells, e.g. bacterial cells, yeast cells, fungi or mammalian cells,
and the vector or nucleic acid can be introduced (transformed) into those

WO 00/09716 PCT/EP99/06022

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ceils stably or transiently by conventional methods, protocols for which can
be found in Sambrook et al. (supra).

Yet a further aspect of the invention is a reagent kit preferably comprising
5 vectors as described above together with support materials for carrying out
the affinity steps. The support materials carry moieties which are capable
of specifically binding the affinity tags, for example, calmodulin-coated resin
in the case of calmodulin binding peptide as the affinity tag or IgG-coated
resins for affinity tags consisting of protein A domains. Additionally, such

10 a kit may comprise buffers and other conventional materials for protein
purification, especially for affinity chromatography. Further, the kit
preferably provides at least one proteolytic agent such as a chemical agent
capable of performing proteolysis or a protease and/or chemical agents such
as chelating agents, wherein the protease is capable of proteolytically

1 5 cleaving the fusion protein. When two proteolytic cleavage sites are used
the kit will preferably contain two proteases.

The following Examples and Figures serve to illustrate the invention and its
practical application, they are, however, not intended to limit the scope of
20 the invention.

Fig. 1 shows a Coomassie stained gel depicting the fractionated

proteins of a yeast RNA-protein complex. Proteins identified by
mass spectrometry are labeled 1-24. Bands 1 , 3, 8-1 1 , 1 6-24

25 were expected in this complex. Bands 2, 4-7 represent

proteins that are likely candidates for true complex component
given their sequence. Bands 12-14 represent potential
contaminants (ribosomal proteins). Band 15 is a trace amount
of TEV protease that remained in this particular preparation.

30 This is not generally the case (see Fig. 2 for example). Bands

4-6 originate from the same gene and might represent
alternative translation products or degradation products. Band

WO 00/09716

Fig. 2

Fig.3

PCT/EP99/06022

- 16 -

16 is a mixture and contains, in addition to a bona fide
complex protein, a contaminating ribosomal protein.

shows a Coomassie stained gel depicting the fractionated
proteins moiety of the yeast U1 snRNP. The 10 specific
proteins were identified by mass spectrometry. Compare wrth
the silver stained gel obtained following a purification using a
cap affinity step and a Ni-NTA column reported by Neubauer
et al., (Proc.Natl.Acad.Sci USA 1997. 94, 385-390). Note in
particular the low level of contaminants in this purification.

shows a Coomassie stained gel in which purified U1 snRNP
has been analysed using either the CBP binding/EGTA eiution
alone (lane 1 ), the Protein A binding/TEV eiution alone (lane 3)
or both steps (lane 7). Arrows on the right point to the U1
snRNP specific proteins. Lanes 2, 4 and 8 show the
background signal obtained using each of the single steps or
the two step procedure from an extract without tagged
protein, demonstrating the requirement for two steps to get a
pure material. Lane 6 is a molecular weight marker. Lane 7 .s
the TEV protease that can also be seen as an abundant
contaminant (even though only the required amount of
protease was used) in lanes 3 and 4. This demonstrates aga.n
the need for a second purification step.

Examples

Example 1

30 Purification of protein complexes from yeast

WO 00/09716 PCT/EP99/06022

- 17 -

A vector encoding a fusion of a yeast protein to the CBP-TEV-Protein A
double tag was constructed using standard methods. The fusion protein is
one subunit of a protein complex of yeast containing 24 subunits in total.
The plasmid was transformed into yeast cells and a 2 L culture of cells
5 expressing the protein was prepared. Proteins were extracted from the
cultured cells using a French press. The complex was purified by binding to
IgG-linked beads, eluting by TEV protease cleavage, binding of the eluted
material on calmodulin containing beads followed by elution with EGTA. All
steps were carried out at 0-4° C, excepted for TEV cleavage.

The first affinity step (IgG step) was performed as follows:
200 jjI of IgG-Sepharose bead suspension (Pharmacia 17-0969-01) were
washed in an Econocolumn with 5 ml of IPP 1 50-lgG buffer (10 mM Tris-CI
pH 8.0, 150 mM NaCI, 0.1% NP40). 10 ml extract, corresponding

1 5 approximately to 2 L of yeast culture, were adjusted to IPP 1 50-lgG buffer
concentrations in Tris-CI pH 8.0, NaCI and NP40. This extract solution was
mixed with the 200 //I of IgG-Sepharose beads and rotated in the
Econocolumn for 2 hours. The unbound fraction was discarded and beads
with bound material were washed first with 30 ml IPP 1 50-lgG buffer

20 followed by 10 ml TEV cleavage buffer (10 mM Tris-CI pH 8.0, 150 mM
NaCI, 0.1% NP40, 0.5 mM EDTA, 1 mM DTT).

The target protein was cleaved and released from the beads as follows. The
washed Econocolumn was filled with 1 ml TEV cleavage buffer and 30 //I
25 TEV protease and rotated in a 1 6°C incubator for 2 hours. The eliiate was
recovered by gravity flow.

The second affinity step (Calmodulin affinity step) was performed as
follows:

30 The previous eluate was mixed with 3 ml of IPP 1 50-Caimodulin binding
buffer (10 mM £-mercaptoethanol, 10 mM Tris-CI pH 8.0, 150 mM NaCI,
1 mM Mg-acetate, 1 mM imidazole, 2 mM CaCI 2 , 0.1% NP40). The

WO 00/09716 PCT/EP99/06022

- 18 -

appropriate amount of CaCl 2 was further added to block the EDTA coming
from the TEV cleavage buffer. This mix was rotated for 1 hour in an
Econocolumn containing 200 //I of Calmodulin beads slurry (Stratagene
214303) previously washed with 5 ml IPP 1 50-Calmodulin binding buffer.

After washing with 30 ml of IPP 1 50-Calmodulin binding buffer, protein
complexes were eluted with 5 successive additions of 200 //I of IPP 1 50-
Calmodulin elution buffer (10 mM jff-mercaptoethanol, 10 niM Tris-CI pH
8.0, 150 mM NaCI, 1 mM Mg-acetate, 1 mM imidazole, 2 mM EGTA, 0.1 %
10 NP40).

Samples were frozen in dry ice and stored at -80°C. Proteins were
concentrated by TCA precipitation (A. Bensadoun and D. Weinstein (1 976),
Anal. Biochem. 70, 241). The proteins were detected by polyacrylamide gel
1 5 electrophoresis with subsequent staining of the gel with Coomassie blue.
The result of the protein purification, a gel of which is depicted in Fig.1
demonstrates that the strategy employed is highly efficient. All the
expected protein subunits which number 24 in this case can be detected.

20 Example 2

The same procedure was used for two other protein or protein-RNA
complexes from yeast where all expected protein subunits were detected
using the method of the invention. Those are the CBC (Cap Binding

25 Complex) and the U1 snRNP. The purified U1 snRNP is depicted in Figures
2 and 3. The CBC complex has been shown to be still active and the purity
was good in all cases. This method is relatively cheap and not very time-
consuming, since it can be done in one day. Concerning the U1 snRNP, it
is noteworthy that Neubauer et al. (Proc. Natl. Acad. Sci. USA 1997, 94,

30 385-390) carried out a purification of the same complex (U1 snRNP)
extracted from 1 6 L of culture. The proteins of the complex of interest were

WO 00/09716 PCT/EP99/06022

- 19 -

then only visible by silver staining and several contaminants were still
observed.

WO 00/09716

- 20-

PCT/EP99/06022

Claims

Method for detecting and/or purifying substances selected from
proteins, biomolecules, complexes of proteins or biomolecules
subunits thereof, cell components, cell organelles and cells
comprising the steps:

(a) providing an expression environment containing one or more
heterologous nucleic acids encoding one or more polypeptides
and/or one or more subunits of a biomolecule complex, the
polypeptides or subunits being fused to at least two different
affinity tags, one of which consists of one or more IgG binding
domains of Staphylococcus protein A,

(b) maintaining the expression environment under conditions that
facilitate expression of the one or more polypeptides or
subunits in a native form as fusion proteins with the affinity
tags,

(O detecting and/or purifying the one or more polypeptides or
subunits by a combination of at least two different affinity
purification steps each comprising binding the one or more
polypeptides or subunits via one affinity tag to a support
material capable of selectively binding one of the affinity tags
and separating the one or more polypeptides or subunits from
the support material after substances not bound to the support
material have been removed.

Method for detecting and/or purifying biomolecule and/or protein
complexes, comprising the steps:

(a) providing an expression environment containing one or more
heterologous nucleic acids encoding at least two subunits of
a biomolecule complex, each being fused to at least one of

- 21 -

different affinity tags, one of which consists of one or more
IgG binding domains of Staphylococcus protein A,

(c) detecting and/or purifying the complex by a combination of at
least two different affinity purification steps each comprising
binding the one or more subunits via one affinity tag to a
support material capable of selectively binding one of the
affinity tags and separating the complex from the support
material after substances not bound to the support material
have been removed.

Method according to claim 1 or 2, wherein between the one or more
polypeptides or subunits and one or more of the affinity tags a
specific proteolytic cleavage site is present in the fusion protein
which facilitates the removal of one or more of the affinity tags.

Method according to claim 3, wherein the specific proteolytic
cleavage site is an enzymatic cleavage site.

Method according to claim 4, wherein the specific proteolytic
cleavage site is the cleavage site for TEV protease NIA.

Method according to claim 3, 4 or 5, wherein the proteolytic
cleavage site is used to cleave the polypeptide or subunit in step (c)
from the IgG binding domain of Staphylococcus protein A bound to
the support material.

PCT/EP99/06022

WO 00/09716

- 22 -

7. Method according to claim 6, wherein the affinity purification of step
(c) comprises:

(i) binding the one or more polypeptides or subunits via the one
or more IgG binding domains of Staphylococcus to a support
material capable of specifically binding the latter, removing
substances not bound to the support material and separating
the one or more polypeptides or subunits from the support
material by cleaving off the IgG binding domains via the
specific proteolytic cleavage site, and
(ii) binding the polypeptide or subunit via another affinity tag to
a second support material capable of specifically binding the
latter, removing substances not bound to the support material
and separating the polypeptide or subunit from the support
material.

Method according to claim 7, wherein step (ii) is carried out before
step (i).

Method according to one of the previous claims, wherein the fusion
protein contains a second specific proteolytic cleavage site for the
removal of one or more of the other affinity tags.

Method according to one of the previous claims, wherein one of the
affinity tags consists of at least one calmodulin binding peptide.

Method according to claim 10, wherein a chemical agent is used to
separate the one or more polypeptides or subunits from the support
material.

12. Fusion protein comprising at least one polypeptide or subunit of a
protein complex fused to at least two different affinity tags, wherein

10.

- 23 -

one of the affinity tags consists of at least one IgG binding domain
of Staphylococcus protein A.

Fusion protein according to claim 1 2, wherein it additionally contains
a specific proteolytic cleavage site.

Nucleic acid coding for a fusion protein according to claim 12 or 13.

Vector comprising a nucleic acid according to claim 14 under the
control of sequences facilitating the expression of a fusion protein
according to claim 12 or 13.

Vector comprising heterologous nucleic acid sequences in form of
one or more cassettes each comprising at least two different affinity
tags one consisting of one or more IgG binding domains of
Staphylococcus aureus protein A, and at least one polynucleotide
linker for the insertion of further nucleic acids.

Vector comprising heterologous nucleic acid sequences in form of
two or more cassettes each comprising at least one of different
affinity tags one consisting of one or more IgG binding domains of
Staphylococcus aureus protein A, and at least one polynucleotide
linker for the insertion of further nucleic acids.

Cell containing a nucleic acid according to claim 14 or a vector
according to claim 15.

Reagent kit comprising a nucleic acid according to claim 14 or a
vector according to claim 1 5, 1 6 or 1 7 for the expression of a fusion
protein according to claim 12 or 13 and support materials each
capable of specifically binding one of the affinity tags.

WO 00/0971 6 PCT/EP99/06022

- 24 -

20. Reagent kit according to claim 1 9 additionally comprising at least
one chemical agent for separating one of the affinity tags from its
support material and/or a specific chemical proteolytic agent and/or
specific protease capable of cleaving the fusion protein.

21. Use of the method according to one of claims 1 to 1 1 for the
detection and/or purification of substances capable of complexing
with the fusion protein.

22. Use of the method according to one of claims 1 to 1 1 for the
detection and/or purification of cells and/or cell organelles expressing
the fusion protein on their surface.

WO 00/09716 PCT/EP99/06022

Figure 1/3

SUBSTITUTE SHEET (RULE 28)

flTiC 3jMJ^' :r ^- r ' BEST AVAILABLE COPY

WO 00/09716

PCT/EP99/06022

Figure 2/3

SUBSTITUTE SHEET (RULE 26)

BEST AVAILABLE COPY

WO 00/09716

PCT/EP99/06022

Figure 3/3

IgG beads - + +

TEV cleavage + +

Calmodulin beads + - +

Extract tag WT tag WT MW Tev tag WT

SUBSTITUTE SHEET (RULE 26)

qpc5t AVAILABLE COPY

INTERNATIONAL SEARCH REPORT

[», aional Application Mo

PCT/EP 99/06022

-^^^^^"cirNlB/U C12N15/62 C12P21/02
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W December 1996 C19?6-12-19>
jy | abstract; claims 1-21; flflwt 5 » samples

page 9, line 10 - Hne_19
SENGER B ET AL.: "MtrlOp functions as a
nuclear Import receptor for the
mRNA-b1nd1ng protein Npl3p
EHBO JOURNAL,

lAuglst 1998 8 (1998-08-01), pages
2196-2207, XP002090021
cited 1n the application
the whole document ^

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1-5,
12-16,
18-20,22
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20 December 1999

Bjwpean Patert OWoe. PB. 5816 Patertlaan 2

Tel. (+31-70) 340-«04aTx. 31 661 eponl.
FfOC (431-70) 340-6016

n PCTrtSASIO (••cand AMI) (AJy 1»2J

patent fantfy mambem are Utod *
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11/01/2000

Authorized offloer

Oderwald, H

page 1 of 2

INTERNATIONAL SEARCH REPORT

lr atonal Application No

PCT/EP 99/06022

C^Continuataon) DOCUMENTS CONSIDERED TO BE RELEVANT

Category * Citation at docvnent, wtth kidcatlon, where appropriate, of the relevant passage*

NOl

ZHENG C ET AL: "A new expression vector
for high level protein production, one
step purification and direct Isotopic
labeling of calmodul1n-b1nd1ng peptide
fusion proteins"
GENE,

vol. 186, no. 1,

20 February 1997 (1997-02-20), page 55-60

XP004054879

the whole document

PANAGIOTIDIS C A ET AL: "pALEX, a

dual -tag prokaryotlc expression vector for

the purification of full-length proteins"

GENE,

vol. 164, no. 1, 1995, page 45-47

XP004041915

the whole document

10,11

Foroi PCT/I8AO10 (oonfruaflon ofsmnd «hMf) (Jury 1002)

page 2 of 2

INTERNATIONAL SEARCH REPORT

PCT/EP 99/06022

Patent document
dted In search report

Pifcflcatton

Patentfamiiy
members)

Pt fcO catl on

WO 9640943

19-12-1996

US
AU
CA
CN
EP
FI
HU
NZ

5821088
6331096
2224172
1192245
0832252

974432
9902004

311902

A
A
A
A
A
A
A
A

13-10-1998
30-12-1996
19-12-1996
02-09-1998

01- 04-1998

02- O2-1998
28-10-1999
23-12-1998

Form PCTBOglO (p«anl M U » mm* (Aiy 1 wa)

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