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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 



Al 



(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) 



EP 



(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. 



AL 


Albania 


ES 


Spain 


LS 


Lesotho 


SI 


Slovenia 


AM 


Armenia 


Fl 


Finland 


LT 


Lithuania 


SK 


Slovakia 


AT 


Austria 


FR 


France 


LU 


Luxembourg 


SN 


Senegal 


AU 


Australia 


GA 


Gabon 


LV 


Latvia 


SZ 


Swaziland 


AZ 


Azerbaijan 


GB 


United Kingdom 


MC 


Monaco 


TD 


Chad 


BA 


Bosnia and Herzegovina 


GE 


Georgia 


MD 


Republic of Moldova 


TG 


Togo 


BB 


Barbados 


GH 


Ghana 


MG 


Madagascar 


TJ 


Tajikistan 


BE 


Belgium 


GN 


Guinea 


MK 


The former Yugoslav 


TM 


Turkmenistan 


BF 


Burkina Paso 


GR 


Greece 




Republic of Macedonia 


TR 


Turkey 


BG 


Bulgaria 


HI) 


Hungary 


ML 


Mali 


TT 


Trinidad and Tobago 


BJ 


Benin 


IE 


Ireland 


MN 


Mongolia 


UA 


Ukraine 


BR 


Brazil 


IL 


Israel 


MR 


Mauritania 


UG 


Uganda 


BY 


Belarus 


IS 


Iceland 


MW 


Malawi 


US 


United States of America 


CA 


Canada 


IT 


Italy 


MX 


Mexico 


UZ 


Uzbekistan 


CF 


Central African Republic 


JP 


Japan 


NE 


Niger 


VN 


Viet Nam 


CG 


Congo 


KE 


Kenya 


NL 


Netherlands 


YU 


Yugoslavia 


CH 


Switzerland 


KG 


Kyrgyzstan 


NO 


Norway 


ZW 


Zimbabwe 


CI 


C6te d'lvohe 


KP 


Democratic People's 


NZ 


New Zealand 






CM 


Cameroon 




Republic of Korea 


PL 


Poland 






CN 


China 


KR 


Republic of Korea 


PT 


Portugal 






CU 


Cuba 


KZ 


Kazakstan 


RO 


Romania 






CZ 


Czech Republic 


LC 


Saint Lucia 


RU 


Russian Federation 






DE 


Germany 


U 


Liechtenstein 


SD 


Sudan 






DK 


Denmark 


LK 


Sri Lanka 


SE 


Sweden 






EE 


Estonia 


LR 


Liberia 


SG 


Singapore 







WO 00/09716 



- 1 - 



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 



- 2 - 



10 



° 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 

- 3 - 

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 

- 4 - 



15 



„ 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: 



25 



s 



WO 00/0971 6 PCT/EP99/06022 

- 5 - 

(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 



10 



15 



20 



25 



PCT/EP99/06022 

WO 00/09716 

- 6 - 

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. 



7 



WO 00/0971 6 PCT/EP99/06022 

- 7 - 

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. 

15 

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 

- 8 - 

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. 



I* 

I 

WO 00/09716 PCT/EP99/06022 

- 9 - 

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. 

5 

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. 

10 

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 



L 



10 



15 



20 



25 



PCT/EP99/06022 

WO 00/09716 

- 10- 

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. 



30 



WO 00/09716 PCT/EP99/06022 

- 11 - 

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. 

20 

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. 



WO 00/0971 6 PCI7EP99/06022 

- 12 - 

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 

- 13 - 

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. 

15 

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 

- 14 - 

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 

- 15 - 

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 



10 

Fig.3 

15 



20 



25 



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. 

10 

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. 

5 

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, 

(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 
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 
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. 



8. 



9. 



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. 



11 



- 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. 



r 

WO 00/09716 PCT/EP99/06022 



Figure 1/3 




r 

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 



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PCT/EP 99/06022 



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



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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) 



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Patent document 
dted In search report 



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members) 



Pt fcO catl on 



WO 9640943 



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FI 
HU 
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01- 04-1998 

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23-12-1998 



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