Affinity Marker comprising a First and a Second Tag and its Use

The present invention relates to an affinity marker comprising a first and a second tag, a protein comprising the affinity marker, a nucleic acid coding for the affinity marker or the protein, a vector comprising the nucleic acid, a cell comprising the nucleic acid or the vector, a method of determining or localizing a nucleic acid sequence or a method of purifying a protein with the affinity marker, the use of the affinity marker for the purification of a protein and/or for determining or localizing a protein or a nucleic acid sequence and a kit comprising the nucleic acid and/or the vector and optionally (a) suitable antibody/ies.

In many areas of industry and research, nowadays pure or even ultrapure products are required, since product quality is often determined by their purity. This applies in particular to proteins produced by biotechnological methods, as these are now finding increasing application in a number of industrial products and processes. They are used for example as medicinal products, for diagnostic or scientific purposes. Furthermore, they are used in many areas of everyday life, for example as diet supplements, as additives in the food industry, for example as baking aids or in cheese-making, but also for example in papermaking, in the hygiene area or in detergents. Ultrapure proteins are in addition required for analytical methods, e.g. for elucidation of structure.

There are already many known methods by which products can be separated from other constituents. As a rule these make use of the differences in physical and chemical properties of the constituents of a sample that is to be purified. Conventional methods of purification and isolation include for example extraction, precipitation, recrystallization, filtration, centrifugation, washing and drying. The separation techniques and principles of adsorption, chromatography or ion exchange are also used. Various column materials are available for chromatographic methods, and make it possible to adapt the purification

process to the particular product that is to be purified, making use of the differences in migration rates of the individual constituents, based for example on charge or hydrophobicity.

Affinity chromatography, in which a product, as a rule a protein, is separated and thus purified on the basis of its affinity for a binding partner, is especially suitable. So-called tags have now been developed, which are for example attached to a protein that is to be purified. The tag possesses an affinity for a particular binding partner; this can be utilized in affinity chromatography. Such tags are in principle of universal application and can be attached to various molecules. In this way it is possible to purify different molecules, especially proteins, with the same method of purification. Thus, ideally, the method does not need to be adapted specifically to the particular product.

Especially for analyzing and purifying proteins, the technique of affinity labeling, i.e. the attachment of a marker or tag to a protein by techniques of molecular biology, is now a frequently used method. In this technique, the primary sequence of any protein is expanded by just a few amino acids by means of recombinant techniques. The presence of a specific binding molecule with high affinity, e.g. an antibody, with a known recognition sequence, is decisive.

A number of (affinity) tags or (affinity) markers are known at present. These are usually divided into 3 classes according to their size: small tags have a maximum of 12 amino acids, medium-sized ones have a maximum of 60 and large ones have more than 60. The small tags include the Arg-tag, the His-tag, the Strep-tag, the Flag-tag, the T7-tag, the V5- peptide-tag and the c-Myc-tag, the medium-sized ones include the S-tag, the HAT-tag, the calmodulin-binding peptide, the chitin-binding peptide and some cellulose-binding domains. The latter can contain up to 189 amino acids and are then regarded, like the GST- and MBP-tag, as large affinity tags.

In order to produce especially pure proteins, so-called double tags or tandem tags were developed. In this case the proteins are purified in two separate chromatography steps, in each case utilizing the affinity of a first and then of a second tag. Examples of such double

or tandem tags are the GST-His-tag (glutathione-S-transferase fused to a polyhistidine- tag), the 6xHis-Strep-tag (6 histidine residues fused to a Strep-tag (see below)), the 6xHis- taglOO-tag (6 histidine residues fused to a 12-amino-acid protein of mammalian MAP- kinase 2), 8xHis-HA-tag (8 histidine residues fused to a haemagglutinin-epitope-tag), His- MBP (His-tag fused to a maltose-binding protein, FLAG-HA-tag (FLAG-tag fused to a hemagglutinin-epitope-tag), and the FLAG-Strep-tag. Most of these tags have been specifically developed for the purification of proteins produced by prokaryotic cells.

Often, however, it is necessary or desirable to purify proteins that were expressed by eukaryotic cells, e.g. if the protein is modified posttranslationally or proteins that are additionally present in the cell, and which bind to the target protein, are to be purified at the same time. The complexity of eukaryotic proteomes makes the purification of proteins that are expressed by eukaryotes challenging, and as a rule means that an individual purification protocol must be established for each protein. As a rule, therefore, the aforementioned double tags cannot be applied directly in eukaryotic systems.

Additionally, affinity tags are also used in the elucidation of nucleic acid sequences and their interaction with proteins. A recently developed technique includes Chromatin ϊrnrnunoprεcipitation (ChIP), a procedure used to determine whether a given protein binds to a specific DNA sequence in a eukaryotic cell or to localize a DNA sequence in a eukaryotic cell.

Therefore, double tags have already been developed for purification of proteins from eukaryotic cells as well. These encompass e.g. FLAG Strep tandem tags. In an experimental setup, the properties of various tags, e.g. HIS, CBP, CYD (covalent yet dissociable NorpD peptide), Strep II, FLAG, HPC (heavy chain of protein C), GST and MBP were compared with respect to their purification properties in various systems (Lichty et al., 2005, Protein Expr Purif 41 : 98-105). The authors recommend, taking into account their results, a combination of 6xHis- and Strep II-tag for double purification.

However, the above methods often require a very high extent of purification capability demanding tags and antibodies binding to each other with high affinity.

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The problem to be solved by the present invention was accordingly to provide an improved affinity marker, which is suitable for the purification of proteins, especially from eukaryotic cells, and/or for use in immunoprecipitation and ChIP techniques. In particular, a problem to be solved by the present invention was to provide an affinity marker for the purification of proteins from (eukaryotic) cells, offering maximum possible yields with preferably highest possible purity of the purified protein. In a preferred embodiment of the invention the affinity marker is intended for use in immunoprecipitation and chromatin immunoprecipitation techniques providing new potentials for improving detection and/or localization of proteins and/or nucleic acids. Further objects of the present invention relate to an affinity marker providing a standard or recovery marker, especially in immunoprecipitation and ChIP techniques.

The object of the present invention was solved by a new affinity marker comprising a first tag comprising a first epitope for an IgG antibody, wherein the first epitope is derived from a cellular protein, and a second tag comprising a second epitope for an artificial antibody.

Accordingly, a first subject of the invention relates to an affinity marker comprising

- a first tag comprising a first epitope for an IgG antibody, wherein the first epitope is derived from a cellular protein; and

- a second tag comprising a second epitope for an artificial antibody.

Surprisingly, it was found that an affinity marker comprising a combination of an epitope for an IgG antibody, wherein the first epitope is derived from a cellular protein, and an artificial antibody is superior to the existing double tag or tandem affinity markers.

The present invention provides a new concept of tandem affinity markers. The affinity marker involves two different separation steps, since two completely different types of epitopes for two different classes of antibodies (IgG and an artificial antibody) are used, which leads to a surprisingly high degree of separation and/or purification. In addition to very high affinities of the respective antibodies for the prevailing epitope, which already provides a quite high degree of separation/purification, the change of purification partners

for each of the epitopes from IgG to an artificial antibody or vice versa provides for an additional purification effect. The diversity of the natures of the antibodies leads to different kinds of impurities in each of the purification steps, which the other purification step compensates for, which in return results in a very high purity at comparably high yields.

As detailed above, one of the epitopes of the affinity marker is an epitope for an IgG antibody. An IgG antibody is a monomeric immunoglobulin, built of two heavy chains and two light chains. Each molecule has two antigen binding sites. In the present invention the affinity marker comprises an epitope for an IgG antibody and is derived from a cellular protein.

"Derived from a cellular protein" in the present context relates to a protein which is identical to or related to a cellular protein, wherein "related to a cellular protein" also refers to a portion of a cellular protein sufficient to be specifically recognized by an IgG antibody. Preferably the portion of the cellular protein encompasses at least 10 consecutive amino acids of that cellular protein, preferably at least 12, more preferably at least 15, still more preferably at least 17 and most preferably at least 20 or 21 consecutive amino acids of the cellular protein. Also encompassed by the term "related to a cellular protein" is a full length cellular protein or a portion of a cellular protein as defined above in which at most about 10 %, more preferably at most about 5 %, still more preferably at most about 1 % of the amino acids have been substituted for another amino acids, more preferably wherein the substitution is a conservative amino acid substitution.

Conservative amino acid substitutions are those that take place within a family of amino acids that are related in their side chains and chemical properties. Examples of such families are amino acids with basic side chains, with acidic side chains, with non-polar aliphatic side chains, with non-polar aromatic side chains, with uncharged polar side chains, with small side chains, with large side chains etc. Examples of conservative amino acid substitutions include, but are not limited to, those listed below:

Original Residue Conservative Substitutions

Ala Ser

Arg Lys

Asn GIn; His

Asp GIu

Cys Ser

GIn Asn

GIu Asp

His Asn; GIn

He Leu, VaI

Leu He; VaI

Lys Arg; GIn; Asn

Met Leu; He

Phe Met; Leu; Tyr

Ser Thr

Thr Ser

Trp Tyr

Tyr Trp; Phe

VaI He; Leu

By "cellular protein" is meant a protein which is produced by a naturally occurring cell. The protein may be present in the cell until e.g. being degraded or otherwise chemically altered or may be released from that cell after being produced e.g. in order to act on or in a different cell.

Examples of such proteins include enzymes, e.g. a oxidoreductase, a transferase, a hydrolyse, a lyase, a isomerase, a ligase, a kinase or a lipase, a dehydrogenase, a reductase, an oxidase, a synthase, a dismutase, a phosphorylase, a transaminase, an esterase, a peptidase, a decarboxylase, a dehydratase, a racemase, an isomerase, an epimerase or a mutase; proteins involved in the signal transduction, such as various kinases and phosphatases, e.g. PKA or PKC, adenylyl cyclase, a tyrosine kinase, transcription factors

such as TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, E2F1 , E2F2, E2F3a, E2F3b, E2F4, E2F5, E2F6, E2F7 or E2F8, factors regulating expression of a gene or PLC; proteins involved in regulation of the cell cycle; proteins for transporting a compound; structural cellular proteins such as actin or tubulin or membrane proteins such as receptors or channels.

In a preferred embodiment the affinity marker of the invention is characterized in that the first epitope is derived from a cellular protein of a eukaryotic cell, preferably a vertebrate cell, more preferably a mammalian cell, still more preferably a human cell, wherein the above definition of "derived from" is to be applied analogously.

In an even more preferred embodiment the affinity marker of the invention is not present in prokaryotic cells.

Yet in another more preferred embodiment of the invention the cellular protein is derived from a protein regulating expression, preferably transcription of a gene, more preferably a protein capable of binding to a sequence directing the transcription of a gene such as a promoter or enhancer, e.g. a transcription factor. Examples of transcription factors are mentioned above.

In an even more preferred embodiment the affinity marker of the invention is characterized in that the cellular protein is a protein capable of binding DNA, optionally a complex of DNA and one or more proteins, more preferably the TATA box binding protein (TBP) - TATA Element Complex.

In a still more preferred embodiment the affinity marker of the invention is characterized in that the cellular protein is derived from negative co-factor 2 (NC2), preferably NC2α. NC2α is also referred to as DrI -associated corepressor. The human amino acid sequence of this factor is available to the public, e.g. at the NCBI (Accession number Q 14919) and has also been published (Goppelt et al., 1996, EMBO J. 15: 3105-31 16). The IgG antibody is preferably directed against an epitope which is located in a portion of NC2α, more

preferably of human NC2α, especially in a prolin-rich portion of (human) NC2α which is located close to the N-terminal region.

In a highly preferred embodiment of the invention the first tag comprises or consists of the sequence:

MSPPTPFLPFASTLPLPPAPP

(SEQ ID NO: 1)

(i.e. methionin plus amino acids 171 to 190 of human NC2α)

However, the tag may differ from the sequence of SEQ ID NO:1 by at most 5, preferably at most 4, more preferably at most 3, still more preferably at most 2, most preferably at most 1 amino acid addition, deletion and/or substitution. In case of substitution one or more conservative amino acid substitution(s) are preferred, wherein the conservative amino acid substitution is as defined above. The one or more amino acid addition(s), deletion(s) and/or substitution(s) may be C-terminally, N-terminally or internally as well as combinations thereof. If more than one amino acid is altered by addition(s), deletion(s), substitution(s) or a combination thereof, the two or more mutations are preferably separated by at least 3, 4 or 5, more preferably at least 10 or 15 unchanged amino acids. Additionally, one or more C-terminal or N-terminal mutation(s)/alteration(s) is/are preferred compared as to one or more internal mutation(s).

In another still more preferred embodiment the affinity marker of the invention is characterized in that the cellular protein is derived from Epstein-Barr virus nuclear antigen (EBNA), preferably EBNAl . The EBNAl protein of Epstein-Barr virus is important for the replication, segregation, and transcriptional activation of latent Epstein-Barr virus genomes. Additionally, it has been implicated in host cell immortalization and avoids proteasomal processing and cell-surface presentation. The human amino acid sequence of this antigen is available to the public, e.g. at the NCBI (Accession number P0321 1) and has also been published (Baer, R. et al., 1984, Nature 310 (5974), 207-21 1). The IgG antibody is preferably directed against an epitope which is located at EBNAl H4.

In a highly preferred embodiment of the invention the first tag comprises or consists of the sequence:

GTGGPADDPGEGSGPADDPGEGSGPADDPGEGSGW (SEQ ID NO:5)

(i.e. sequence of EBNAl)

However, the tag may differ from the sequence of SEQ ID NO:5 by at most 5, preferably at most 4, more preferably at most 3, still more preferably at most 2, most preferably at most 1 amino acid addition, deletion and/or substitution. In case of substitution one or more conservative amino acid substitution(s) are preferred, wherein the conservative amino acid substitution is as defined above. The one or more amino acid addition(s), deletion(s) and/or substitution(s) may be C-terminally, N-terminally or internally as well as combinations thereof. If more than one amino acid is altered by addition(s), deletion(s), substitution(s) or a combination thereof, the two or more mutations are preferably separated by at least 3, 4 or 5, more preferably at least 10 or 15 unchanged amino acids. Additionally, one or more C-terminal or N-terminal mutation(s)/alteration(s) is/are preferred compared as to one or more internal mutation(s).

The affinity marker is further characterized by the presence of a second tag comprising a second epitope for an artificial antibody (synAB). Each of the tags comprises or consists of the respective epitope; the tags may encompass additional elements, in general it consists of the epitope and optionally one or more additional amino acids.

An artificial antibody in the context of the present invention is an antibody whose structure is different from naturally occurring antibodies such as IgA, IgD, IgE IgG or IgM, the structure of which is well known and characterized and is based on a Y shape. Artificial antibodies are in general characterized by a high affinity binding to their epitopes as compared to naturally occurring or traditional antibodies, since their structure has been adapted and improved for this purpose (increasing affinity).

The artificial antibody may be a single chain antibody, which is smaller in size than the naturally occurring antibodies. The non-natural antibody format of single chain antibodies (SCAs or scFvs) combines essentially only the antigen-binding regions of antibodies on a single stably-folded polypeptide chain. As such, single chain antibodies are of considerably smaller size than classical immunoglobulins but retain the antigen-specific binding properties of antibodies. Consequently, SCAs provide attributes which traditional antibodies cannot deliver. A major obstacle of methods employing traditional antibodies is the non-specific background generated by both the binding and non-binding regions of the intact antibodies. However, protein fragments consisting of the minimal binding subunit of antibodies, i.e. single chain antibodies, have excellent binding specificity and affinity for their epitopes. In contrast to traditional antibodies, single chain antibodies lack the non- binding regions, can be selected in the company of competing antigens, and therefore have potential for higher specificity/sensitivity in separation and purification methods. The production of a scFv directed against GCN4 is exemplified in Zahnd et al, 2004, J Biol Chem. 279: 18870-18877.

Single chain antibodies and their production are disclosed in a series of patents and patent applications including e.g. EP 0281604, US 4,946,778, US 5,260,203, US 5,455,030, US 5,518,889, US 5,534,621 , US 4,704,692, US 5,658,763, US 5,631 ,158, US 5,733,782, EP 0318554, EP 0623679 and US 6,103,889.

Another example of an artificial antibody is a (designed) ankyrin repeat protein. These proteins are disclosed in WO 02/20565. In general the ankyrin repeat protein encompasses repeat domains between capping repeats, which are special terminal repeats of ankyrin repeats. Optional elements are linkers to join further domains such as a further series of repeat domains or a non-repeat domain. Examples of typical ankyrin repeat sequence motifs include without limitation:

- DxxGxTPLHLAaxx-iddddb±±t±±GpxpaVpxLLpxGA±±±±±DVNAx, wherein "x" denotes any amino acid, "±"denotes any amino acid or a deletion, "a" denotes an amino acid with an apolar side chain, and "p" denotes a residue with a polar side chain, DxxGxTPLHLAxxxGxxxVVxLLLxxGADVNAx, wherein "x" denotes any amino acid

DxxGxTPLHLAxxxGxxxlVxVLLxxGADVNAx, wherein "x" denotes any amino acid and

- Dl IGl TPLHLAAl IGHLEI VEVLLK2GAD VNAl , wherein 1 represents an amino acid residue selected from the group: A, D, E, F, H, I, K, L, M, N, Q, R _: S, T, V ₉ W and Y; wherein 2 represents an amino acid residue selected from the group: H, N and Y.

The production and selection of a high affinity designed ankyrin repeat protein is disclosed in Binz et al., 2004, Nature Biotechnology 22: 575-582.

In a preferred embodiment of the invention the second epitope is derived from a protein which is not present in a human cell, preferably a mammalian cell, more preferably a vertebrate cell, still more preferably an animal cell, wherein "derived from" has the meaning as defined above.

In another preferred embodiment of the invention the second epitope is derived from a protein of a fungal cell such as a cell of Rhizomucor, Saccharomyces, Schizoaccharomyces or Aspergillus, preferably a yeast cell, more preferably from Saccharomyces cerevisiae.

In a preferred embodiment of the invention the protein is or is derived from a transcription factor, preferably a transcription factor responsible for the activation of one or more genes required for amino acid or for purine biosynthesis.

In a even more preferred embodiment of the invention the second epitope is derived from GCN4, particularly Saccharomyces cerevisiae GCN4, the sequence of which is public available at NCBI (Accession number AAL09032) and has been published by Czerwinski et al., 2002, Transfusion 42: 257-264.

The epitope may differ from the above sequence as published (see Czerwinski et al., supra) by at most 5, preferably at most 4, more preferably at most 3, still more preferably at most 2, most preferably at most 1 amino acid addition, deletion and/or substitution. In case of substitution one or more conservative amino acid substitution(s) are preferred, wherein the conservative amino acid substitution is as defined above. The one or more amino acid

addition(s), deletion(s) and/or substitution(s) may be C-terminally, N-terminally or internally as well as combinations thereof. If more than one amino acid is altered by addition(s), deletion(s), substitution(s) or a combination thereof, the two or more mutations are preferably separated by at least 3, 4 or 5, more preferably at least 10 or 15 unchanged amino acids. Additionally, one or more C-terminal or N-terminal mutation(s)/alteration(s) is/are preferred compared to one or more internal mutation(s).

An especially preferred second tag comprises or consists of the sequence

RMKQLEPKVEELLPKNYHLENEVARLKKLVGER (SEQ ID NO:2),

which differs from the sequence as published (see Czerwinski et al., supra) by deletion of the 2 C-terminal amino acids (T and S) and substitution of amino acid 7 and 14, wherein D and S, respectively, are replaced with P.

As detailed above, the second epitope may be an epitope for an ankyrin repeat protein as defined above. An especially preferred second tag encompassing an epitope for an ankyrin repeat protein comprises or consists of the sequence of maltose-binding protein (MBP). Particularly, the nucleic acid sequence encoding the tag may be

AGCTTGCGGCCATGAAAACTGAAGAAGGTAAACTGGTAATCTGGATTAA CGGCGATAAAGGCTATAACGGTCTCGCTGAAGTCGGTAAGAAATTCGAG AAAGATACCGGAATTAAAGTCACCGTTGAGCATCCGGATAAACTGGAAG AGAAATTCCCACAGGTTGCGGCAACTGGCGATGGCCCTGACATTATCTTC

TGGGCACACGACCGCTTTGGTGGCTACGCTCAATCTGGCCTGTTGGCTGA AATCACCCCGGACAAAGCGTTCCAGGACAAGCTGTATCCGTTTACCTGGG ATGCCGTACGTTACAACGGCAAGCTGATTGCTTACCCGATCGCTGTTGAA GCGTTATCGCTGATTTATAACAAAGATCTGCTGCCGAACCCGCCAAAAAC CTGGGAAGAGATCCCGGCGCTGGATAAAGAACTGAAAGCGAAAGGTAA

GAGCGCGCTGATGTTCAACCTGCAAGAACCGTACTTCACCTGGCCGCTGA TTGCTGCTGACGGGGGTTATGCGTTCAAGTATGAAAACGGCAAGTACGA

CATTAAAGACGTGGGCGTGGATAACGCTGGCGCGAAAGCGGGTCTGACC TTCCTGGTTGACCTGATTAAAAACAAACACATGAATGCAGACACCGATTA CTCCATCGCAGAAGCTGCCTTTAATAAAGGCGAAACAGCGATGACCATC AACGGCCCGTGGGCATGGTCCAACATCGACACCAGCAAAGTGAATTATG GTGTAACGGTACTGCCGACCTTCAAGGGTCAACCATCCAAACCGTTCGTT

GGCGTGCTGAGCGCAGGTATTAACGCCGCCAGTCCGAACAAAGAGCTGG CAAAAGAGTTCCTCGAAAACTATCTGCTGACTGATGAAGGTCTGGAAGC GGTTAATAAAGACAAACCGCTGGGTGCCGTAGCGCTGAAGTCTTACGAG GAAGAGTTGGCGAAAGATCCACGTATTGCCGCCACTATGGAAAACGCCC AGAAAGGTGAAATCATGCCGAACATCCCGCAGATGTCCGCTTTCTGGTAT

GCCGTGCGTACTGCGGTGATCAACGCCGCCAGCGGTCGTCAGACTGTCGA TGAAGCCCTGAAAGACGCGCAGACTGGATC (SEQ ID NO:3)

However, in another embodiment the second tag may differ from the sequence of SEQ ID NO:2 or the sequence encoded by SEQ ID NO: 3 by at most 10, preferably at most 5, more preferably at most 3, still more preferably at most 2, most preferably at most 1 amino acid addition, deletion and/or substitution. In case of substitution one or more conservative amino acid sυbstitution(s) are preferred, wherein the conservative amino acid substitution is as defined above. Alternatively substitutions replacing any amino acid with prolin are also preferred. The one or more amino acid addition(s), deletion(s) and/or substitution(s) may be C-terminally, N-terminally or internally as well as combinations thereof. If more than one amino acid is altered by addition(s), deletion(s), substitution(s) or a combination thereof, the two or more mutations are preferably separated by at least 3, 4 or 5, more preferably at least 6, 7, 8, 9 or 10 unchanged amino acids. Additionally, one or more C- terminal or N-terminal mutation(s)/alteration(s) is/are preferred as compared to one or more internal mutation(s).

For the development of antibodies against GCN4 ribosome display was applied for affinity selection of antibody single-chain fragments (scFv) from a diverse library generated from mice immunized with a variant peptide of the transcription factor GCN4 (7Pl 4P) dimerization domain (Hanes J et al., 1998, Proc Natl Acad Sci U S A. 24; 95: 14130-5. The

best scFv, which may also be used in the context of the present invention, had a dissociation constant of 4 ± 1 x 10 ^' " M, measured in solution.

In a further even more preferred embodiment of the invention the second epitope is derived from a prion protein (PrP or PrP epitope), particularly a mammalian prion, especially bovine prion the sequence of which is public available at NCBI (Accession number NP_851358) and has been published by Espinosa et al, 2007, J. Virol. 81 (2), 835-843 (see also B Luginbuehl et al., 2006, J. MoI. Biol. 363;75-97).

The epitope may differ from the above sequence as published (see Espinosa et al., supra) by at most 5, preferably at most 4, more preferably at most 3, still more preferably at most 2, most preferably at most 1 amino acid addition, deletion and/or substitution. In case of substitution one or more conservative amino acid substitution(s) are preferred, wherein the conservative amino acid substitution is as defined above. The one or more amino acid addition(s), deletion(s) and/or substitution(s) may be C-terminally, N-terminally or internally as well as combinations thereof. If more than one amino acid is altered by addition(s), deletion(s), substitution(s) or a combination thereof, the two or more mutations are preferably separated by at least 3, 4 or 5, more preferably at least 10 or 15 unchanged amino acids. Additionally, one or more C-terminal or N-terminal mutation(s)/alteration(s) is/are preferred compared to one or more internal mutation(s).

An especially preferred second tag comprises or consists of the sequence

GQGGGTHGQWNKPSKP (SEQ ID NO:6)

(i.e. amino acids 90 to 105 of bovine prion (BoPrP (90-105))

Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative prion disease affecting cattle that is transmissible to humans, manifesting as a variant of Creutzfeldt- Jakob disease (vCJD) likely following the consumption of meat contaminated with BSE prions. High-affinity antibodies able to detect even small amounts of the disease-associated PrP conformer (PrPSc) have been described (B Luginbuehl et al., 2006, J. MoI. Biol.

363;75-97), wherein the single-chain Fv antibody fragment have a binding affinity of 1 pM to a peptide derived from the BoPrP (90-105). These antibodies may be used in the context of the present invention.

In an even more preferred embodiment of the invention, the MBP epitope may be combined with an NC2 epitope, more preferably an NC2 epitope as defined above or with an EBNA epitope, more preferably an EBNAl epitope as defined above. A highly preferred affinity marker may be composed as follows:

Optionally linker/cloning site - Tag with MBP epitope (second epitope) - linker/cloning site - Tag with IgG epitope (first epitope) - optionally linker/cloning site, or optionally linker/cloning site - Tag with epitope for artificial antibody (second epitope) - linker/cloning site - Tag with NC2 epitope (first epitope) - optionally linker/cloning site, particularly

Optionally linker/cloning site - Tag with MBP epitope (second epitope) - linker/cloning site - Tag with NC2 epitope (first epitope) - optionally linker/cloning site especially

Tag with MBP epitope (second epitope) - linker/cloning site - Tag with NC2 epitope

(first epitope) or optionally linker/cloning site - Tag with epitope for artificial antibody (second epitope) - linker/cloning site - Tag with EBNA epitope (first epitope) - optionally linker/cloning site, particularly optionally linker/cloning site - Tag with MBP epitope (second epitope) - linker/cloning site - Tag with EBNA epitope (first epitope) - optionally linker/cloning site especially

Tag with MBP epitope (second epitope) - linker/cloning site - Tag with EBNA epitope (first epitope) or vice versa.

In a further even more preferred embodiment of the invention, the GCN4 epitope may be combined with an NC2 epitope, more preferably an NC2 epitope as defined above or with an EBNA epitope, more preferably an EBNAl epitope as defined above. A highly preferred affinity marker may be composed as follows:

Optionally linker/cloning site - Tag with GCN4 epitope (second epitope) - linker/cloning site - Tag with IgG epitope (first epitope) - optionally linker/cloning site, or optionally linker/cloning site - Tag with epitope for artificial antibody (second epitope) - linker/cloning site - Tag with NC2 epitope (first epitope) - optionally linker/cloning site, particularly optionally linker/cloning site - Tag with GCN4 epitope (second epitope) - linker/cloning site - Tag with NC2 epitope (first epitope) - optionally linker/cloning site especially

Tag with GCN4 epitope (second epitope) - linker/cloning site - Tag with NC2 epitope (first epitope) or optionally linker/cloning site - Tag with epitope for artificial antibody (second epitope) - linker/cloning site - Tag with EBNA epitope (first epitope) - optionally linker/cloning site, particularly optionally linker/cloning site - Tag with GCN4 epitope (second epitope) - linker/cloning site - Tag with EBNA epitope (first epitope) - optionally linker/cloning site especially

Tag with GCN4 epitope (second epitope) - linker/cloning site - Tag with EBNA epitope (first epitope) or vice versa.

In a further even more preferred embodiment of the invention, the PrP epitope may be combined with an NC2 epitope, more preferably an NC2 epitope as defined above or with an EBNA epitope, more preferably an EBNAl epitope as defined above. A highly preferred affinity marker may be composed as follows:

Optionally linker/cloning site - Tag with PrP epitope (second epitope) - linker/cloning site - Tag with IgG epitope (first epitope) - optionally linker/cloning site, or optionally linker/cloning site - Tag with epitope for artificial antibody (second epitope) - linker/cloning site - Tag with NC2 epitope (first epitope) - optionally linker/cloning site, particularly optionally linker/cloning site - Tag with PrP epitope (second epitope) - linker/cloning site - Tag with NC2 epitope (first epitope) - optionally linker/cloning site especially

Tag with PrP epitope (second epitope) - linker/cloning site - Tag with NC2 epitope

(first epitope) or optionally linker/cloning site - Tag with epitope for artificial antibody (second epitope) - linker/cloning site - Tag with EBNA epitope (first epitope) - optionally linker/cloning site, particularly optionally linker/cloning site - Tag with PrP epitope (second epitope) - linker/cloning site - Tag with EBNA epitope (first epitope) - optionally linker/cloning site especially

Tag with PrP epitope (second epitope) - linker/cloning site - Tag with EBNA epitope (first epitope) or vice versa.

Particularly, the affinity marker of the invention may be composed as shown in the following illustration:

Within the affinity marker both tags may be directly joined to each other or may be joined by a linker. The linker can be any suitable linker, especially a linker of amino acid residues. Preferred linkers are short peptide linkers consisting of 1, 2, 3, 4. 5, 6, 7, 8, 9 or 10 amino acids, preferably 1 , 2, 3, 4 or 5 amino acids, more preferably 1, 2 or 3 amino acids. Preferably each of these amino acids is GIy or Ser or combinations thereof. A particular preferred linker comprises or consists of the amino acid sequence GIy Ser GIy or GIy Ser or GIy Ala GIy.

In a preferred embodiment of the invention the affinity marker comprises or consists of the tags of SEQ ID NO:1 and SEQ ID NO:2, more preferably joined by a linker, especially GIy Ser GIy or GIy Ser.

In another preferred embodiment of the invention the affinity marker comprises or consists of the tags of SEQ ID NO: 1 , SEQ ID NO:2 and SEQ ID NO:6, optionally joined by one or more linkers.

In a preferred embodiment of the invention the affinity marker comprises or consists of the tags of SEQ ID NO: 1 and SEQ ID NO:6, more preferably joined by a linker, especially GIy Ser.

Highly preferred affinity markers of the invention comprise or consist of one of the following sequences:

NC2-GCN4 with methionine MSPPTPFLPFASTLPLPPAPPGSGRMKQLEPKVEELLPKNYHLENEVARLKKLVGE R (SEQ ID NO:4).

NC2-EBNA-PrP SPPTPFLPFASTLPLPP APP-GTGGP ADDPGEGSGPADDPGEGSGP ADDPGEGSG W- GQGGTHGQWNKPSKP (SEQ ID NO:7)

NC2-GCN4 without methionine SPPTPFLPFASTLPLPPAPP-GS-GRMKQLEPKVEELLPKNYHLENEVARLKKLVGER (SEQ ID NO:8)

NC2-PrP

MSPPTPFLPFASTLPLPPAPP-GS-GOGGGTHGQWNKPSKP (SEQ ID NO:9)

In one embodiment of the invention it may be necessary or desirable to cleave off at least one of the two tags of a tagged protein. In this case a cleavage site, for example a cleavage site for an enzyme, may be present. The cleavage site could for example be a protease cleavage site. Examples of proteases are chymotrypsin. trypsin, elastase, and plasmin; the corresponding cleavage sites are known to a person skilled in the art. Since the molecule tagged is a protein, specific proteases, especially proteases from viruses that normally attack plants, are preferred. Examples of suitable specific proteases are thrombin, factor Xa, Igase, TEV-protease from the "Tobacco Etch Virus", the protease PreScission (Human Rhinovirus 3C Protease), enterokinase or Kex2. TEV-protease and PreScission are especially preferred.

The affinity marker according to the present invention can be produced for example by chemical synthesis in a known manner, e.g. by solid phase synthesis of linear peptide building blocks (SPPS) according to Merrifield using suitable protecting groups such as Fmoc. Individual components can also be joined together via peptide bonds. For this purpose it is possible to use e.g. amino acid coupling by activation of the carboxylic acid using for example 1 -hydroxy- 1 H-benzotriazole (HOBt) and carbodiimide (e.g. EDC).

Another subject of the invention relates to a protein comprising the affinity marker of the invention, wherein the affinity marker may be any of the affinity markers as defined above. A protein according to the present invention is a polymer composed of amino acids, with the individual amino acids joined together by peptide bonds to form chains. The length of the amino acid chains can be more than 10000 amino acids, and is preferably more than 50 or more than 100 amino acids. The affinity marker that is bound to the protein to be purified or used in the methods of the invention is produced in a suitable manner by the techniques of genetic engineering using the corresponding nucleic acid and/or a suitable vector. These techniques are well known to a person skilled in the art and are described in more detail below. The protein contains both tags, optionally a cleavage site, and the protein to be purified (protein sample).

The arrangement of the individual components in the protein with affinity marker, the underlying nucleic acid or the underlying vector can be as follows, for example:

First tag-protein-Second tag, Second tag-protein- First tag, First tag-Second tag -protein, Second tag-First tag -protein, Protein-First tag-Second tag or Protein-Second tag-First tag.

A further subject of the present invention is a nucleic acid that codes for a protein containing the affinity marker according to the invention. The nucleic acid may be, for example, an RNA or DNA. These can be used for example for production of the protein,

labelled with the affinity marker that is to be purified. For this, the nucleic acid can be inserted in a cell, especially a eukaryotic cell, and expressed in the cell. Methods for the insertion and expression of nucleic acids in cells are generally familiar to a person skilled in the art. A nucleic acid encoding an affinity marker of the invention and comprising the sequence of SEQ ID NO:3 or coding for any of the sequences of SEQ ID NO: 1, 2 or 4 to 9 is especially preferred.

Vectors are usually used for the production of proteins, especially of recombinant proteins. Accordingly, yet another subject of the present invention is a vector containing a nucleic acid according to the invention. These vectors are then inserted in a target cell and the target cell is cultivated. With selection of a suitable vector, the target cell produces the desired protein. A great many vectors are known in the prior art. As a rule the vector is selected in relation to the target cell, in order to achieve expression that is as efficient as possible. In addition to the nucleic acid for the affinity marker optionally in combination with that for the protein of interest, the vector contains e.g. a suitable promoter, enhancer, selection marker and/or a suitable signal sequence, which for example causes the protein to be secreted into the medium, and optionally suitable cleavage sites for e.g. restriction enzymes. The constituents of vectors are familiar to a person skilled in the art, who will be able to select them in relation to the particular target cell. The restriction enzyme cleavage sites make it possible for nucleic acids that code for various proteins of interest to be incorporated in the vector and removed simply and quickly. Depending on what side the affinity marker is to be bound to the protein subsequently, they can be located 5' or 3' from the nucleic acid that codes for the affinity marker, or between the nucleic acid segments encoded by the first and second tag.

The protein contains the components first tag and second tag plus the protein to be purified or intended for the methods of the invention, each of which are as defined above and can also be arranged thus. Additionally, there may be for example a linker between the two domains, one or more cleavage sites or a signal sequence. The individual components are arranged in the protein as explained above. A person skilled in the art knows how to derive a nucleic acid sequence from a protein sequence, taking into account the genetic code. He also knows how to produce such a nucleic acid sequence using standard techniques of

molecular biology. This can be accomplished for example by the use and combination of existing sequences using restriction enzymes. The nucleic acid suitably also contains further elements e.g. a promoter, enhancer, a transcription start and stop signal and a translation start signal.

The nucleic acid thus produced will then, optionally by means of a vector, be inserted and expressed in a target cell. Accordingly, a further subject of the present invention is a cell containing a nucleic acid according to the invention or a vector according to the invention. The target cell can be any suitable cell, especially a eukaryotic cell, for example a fungal, plant or animal cell. Cell lines of these cells are also included. Preferably it is a mammalian cell, especially a human cell or cell line. Examples of such cells are HEK 293 cells, CHO cells, HeLa cells, CaCo cells, Raji cells or NIH 3T3 cells. Suitable vectors can be used for transfection of the cells. Examples of vectors are pBR322, the pUC series, pBluescript, pTZ, pSP, pREP4 and pGEM. The components of the nucleic acid or of the vector are selected in such a way that the nucleic acid is expressed and the target protein (affinity marker and protein of interest) is produced by the target cell. The cells are cultivated until a sufficient amount of target protein has been produced. Expression and cultivation may e.g. be performed as described in Examples 1 or 2.

Then the protein can be isolated. If a sufficient amount of the target protein has been secreted into the medium, work can continue with this. Otherwise it may be necessary to disrupt the cells. This can be effected for example by lysis of the cells e.g. by means of ultrasound or hypotonic medium. To remove insoluble components, the sample obtained can for example be centrifuged, especially at 10000 x g to 15000 x g, and the supernatant obtained can be used further for the method of purification according to the invention (see below).

Another subject of the invention relates to the use of the affinity marker of the invention for immunoprecipitation or chromatin immunoprecipitation (ChIP).

Immunoprecipitation (IP) is one of the most widely used immunochemical techniques. Immunoprecipitation followed by e.g. SDS-PAGE and immunoblotting, is routinely used

e.g. to study protein/protein interactions, to determine specific enzymatic activity or to determine the presence and quantity of proteins. The IP technique also enables the detection of rare proteins which otherwise would be difficult to detect since they can be concentrated up to 10,000-fold by immunoprecipitation.

In the IP method, the protein from the cell or tissue homogenate is precipitated in an appropriate buffer by means of an immune complex which includes the binding of a primary antibody to its epitope and the binding of a Protein A-, G-, or L-agarose conjugate or a secondary antibody-agarose conjugate. The choice of agarose conjugate depends on the species origin and isotype of the primary antibody. The methods described are comparable and the choice of method depends on the specific antigen-antibody system. In general, methods of performing immunoprecipitation are well known to the skilled practitioner and can be easily adapted when employing the affinity marker of the invention. However, immunoprecipitation may also be performed as described in Example 3.

Chromatin immunoprecipitation (ChIP) is a method to determine whether proteins including (but not limited to) transcription factors bind to a particular region on the endogenous chromatin or on the DNA of living cells or tissues, wherein this test is in general performed using living cells.

ChIP involves the immunoprecipitation of protein/DNA complexes that have been stabilized via cross-linking. Traditionally, transcription factor and gene promoter activity has been analyzed using reporter gene assays, EMSA, Western blot and DNA microarrays. Although these methods have led to significant advances in the scientific understanding of transcription, they cannot be used to demonstrate that a particular protein is bound to a specific, native DNA sequence in living cells. ChIP offers a versatile solution by combining the specificity of immunoprecipitation, the sensitivity of PCR and the screening power of array profiling, all in a single assay.

The principle underpinning this assay is that DNA-bound proteins (including transcription factors) in living cells can be cross-linked to the chromatin on which they are situated. This is usually accomplished by e.g. a gentle formaldehyde fixation. Following fixation, the

cells are lysed, if living cells are used, and the DNA is broken into pieces such as pieces of e.g. 0.2-1 kb in length by e.g. sonication. Once the protein is immobilized on the chromatin and the chromatin is fragmented, whole protein-DNA complexes can be immuno- precipitated using an antibody specific for the protein in question. The DNA from the isolated protein/DNA fraction can then be purified. The identity of the DNA fragments isolated in complex with the protein of interest can then be determined by PCR using primers specific for the DNA regions that the protein in question is hypothesized to bind. Alternatively, when one wants to find where the protein binds across the whole genome, a DNA microarray can be used (ChIP on chip or ChIP-chip) allowing for the characterization of the cistrome. However, the ChIP requires high affinity antibodies having a specificity which are difficult and expensive to produce for each protein of interest. Therefore, affinity markers are conventionally used and linked to the protein of interest (in other words the protein of interest is labeled with an affinity marker) and used for purification or detection reason.

In general ChIP is performed on intact cells, which express the protein of interest linked to the affinity marker of the invention. First, cells may be fixed with formaldehyde, which cross-links and therefore preserves protein/DNA interactions. DNA is then sonicated into small uniform fragments and the DNA/protein complexes are immunoprecipitated using an antibody directed against the DNA-binding protein of interest with is tagged with the affinity marker of the invention. Following immunoprecipitation, cross-linking is reversed, proteins are removed e.g. by Proteinase K treatment and the DNA is cleaned up using the included DNA purification columns. The DNA is then screened to determine which genes were bound by the protein of interest. The versatility of ChIP means that screening can be done using generalized hybridization or a more targeted PCR-based approach.

For successful ChIP, it may be necessary to shear the chromatin to fragments, e.g. 200- 1000 bp fragments. This has traditionally been performed by subjecting the isolated chromatin to different pulses of sonication (e.g. as detailed in the Examples). Alternatively a ,,Enzymatic Shearing Kit" may be used which shears DNA into fragments.

Thereafter. cells may be fixed (e.g for 10 minutes with 1% formaldehyde or as detailed in the Examples) and then chromatin may be sheared as described above (e.g as detailed in the Examples or using a commercially available ,,Enzymatic Shearing Kit"). The sheared and/or unsheared chromatin samples may be subjected to cross-link reversal, treated with Proteinase K, phenol/chloroform extracted and precipitated using the second epitope and the artificial antibody. Samples may be separated by a second affinity purification using the first epitop and the corresponding antibody using for example by electrophoresis e.g. through a 1% agarose gel. Positive and negative controls may be included into the assay. After separation by electrophoresis the obtained sequences may be analyses e.g. by PCR using suitable primers.

In further embodiment the present invention relates to ChIP-chip technology employing the synthetic antibodies with superior biophysical properties as defined above. Conceptually immunoglobulin epitopes are combined with synthetic, in vitro evolved protein scaffolds that recognize defined unique peptides. The goal is e.g. to express rare proteins in mammalian cells fused to these tags, to control expression appropriately and to then isolate complexes and immunoprecipitate the proteins bound to chromatin. This enhances the sensitivity for orders of magnitudes and has the potential to ChIP technology to broad biological and medical applications.

The ChIP-chip technology for the identification or detection of proteins may be carried out as follows:

1. The expression and purification of the synthetic antibody (sAB or synAB)).

2. Vector construction for expression of the Portein of interest tagged with an affinity marker of the invention, such as GCN4 or MBP or prion-tagged interested proteins

3. Transfection of cells with the vector construction and optionally generation of stable cell lines

4. Immobilization of the sAB on suitable solid surface.

5. ChIP experiment.

Accordingly, another subject of the present invention relates to a method of determining or localizing a nucleic acid sequence comprising a) incubating a protein comprising the affinity marker of the invention with a nucleic acid under conditions allowing the formation of a protein nucleic acid complex; b) optionally producing fragments of the nucleic acid of the complex of step a); c) purifying the complex of step a) or b) by means of affinity purification using an antibody against the second epitope; and d) determining and/or localizing the nucleic acid sequence bound to the protein, wherein the first epitope of the affinity marker is used for pre-purification of the protein nucleic acid complex of step a) or b) prior to step c) and/or is used for standardization.

As detailed above, the first epitope is derived from a cellular protein or identical to a cellular protein. Accordingly, the ChIP may encompass two affinity purification steps, wherein the complex is first purified using the first epitope. The resulting purified solution encompasses the complex of tagged protein and DNA as well as the cellular protein produced by the cell due to its normal function. The amount of both may be determined before and after this first purification step. Thereafter, the complex may be purified using the second tag. Accordingly, the cellular protein is removed. The amount of complex may be determined again, wherein the difference of both amounts allows for standardization. In a similar manner the affinity marker may be used as recovery marker.

Each of the steps of the ChIP assay may independently be carried out e.g. as described in Example 4.

A further subject of the present invention is a method for the purification of a protein, especially expressed in a (eukaryotic) cell, preferably a mammalian cell, comprising the steps: a) providing the protein according to the invention; b) purifying the protein of step a) by means of affinity purification using an IgG antibody; and c) purifying the purified protein from step b) by means of affinity purification using an artificial antibody.

Alternatively, the following method can also be used: a) providing the protein according to the invention; b) purifying the protein of step a) by means of affinity purification using an artificial antibody; and c) purifying the purified protein from step b) by means of affinity purification using an IgG antibody.

The sample containing the protein labeled with the affinity marker can be obtained as described above, for example using genetic engineering by expression in a cell, especially a eukaryotic cell.

The sample, obtained as described above, is purified by tandem affinity purification using the first tag and the second tag and the respective antibody (IgG or artificial antibody). Affinity purification is a special form of adsorption purification, in which there are, on a carrier, groups (binding partners) with high affinity and therefore high binding strength to one of the two domains, so that these can be adsorbed preferentially and thus separated from other substances. Purification can be carried out either first via the first tag and then via the second tag. or vice versa. Purification takes place by specific binding to a suitable binding partner. The binding partner is preferably bound to a solid phase. The solid phase can be usual carrier materials, for example Sepharose, Superflow, Macroprep, POROS 20 or POROS 50. Separation is then carried out for example chromatographically, e.g. by gravity, HPLC or FPLC. Alternatively, the binding partner can also be bound to beads, especially magnetic beads. After adding the beads to the sample, binding takes place between the particular domain and the corresponding binding partner. The suspension can then be centrifuged for example, so that the labeled molecule sediments with the bead, and other components remain in the supernatant, from where they can be removed. Alternatively, the suspension is separated utilizing the magnetic properties of the beads. In one embodiment, the suspension is applied to a column, which is located in a magnetic field. As the magnetic beads and the molecule bound to them are retained in the magnetic field, other constituents of the sample can be washed out in several washing operations. The protein of interest can then for example be washed from the beads using a suitable

elution buffer, or can be separated from the beads by enzymatic cleavage e.g. at the cleavage site between the two tags.

The first purification of the protein (step b)) takes place by a) binding of the first tag (or the second tag) to the respective binding partner (see above) in suitable conditions, b) optionally washing and c) detachment of the protein from the binding partner. The latter can either be effected by altering the conditions, so that the changed conditions no longer permit binding between affinity marker and binding partner (e.g. alteration of the pH value or the ionic strength), or by separating the molecule from the domain bound to the binding partner. Separation can be effected by cleavage of the bond between tag and binding partner, e.g. by chemical means or using specific enzymes, as was described in detail above. Alternatively, it is also possible to use specific competitors, which are added in excess.

This is followed by the second purification of the protein (step c)) by a) binding of the second tag (or the first tag), i.e. the tag that was not used in the first purification step, to the respective binding partner in suitable conditions, b) optionally washing and c) detachment of the tag from the binding partner, where these steps can be of the same form as those described for the first purification step.

The binding partner is the respective antibody as detailed above. The IgG antibody used can either be a polyclonal or preferably monoclonal antibody produced by methods known to a person skilled in the art or an antibody known from the prior art. In this case elution of the bound protein can be carried out for example with the competitors such as synthetic peptides.

Preferably, the artificial antibody used in the methods of the present invention is a single chain antibody or an ankyrin repeat protein as defined above. More preferably the single chain antibody is the scFv antibody 52SR4 (Zahnd et ah, supra), which may be used if the second epitope or tag consist of or comprises GCN4, preferably GCN4(7P14P) (SEQ ID NO:2).

The antibodies of the present invention are particularly preferred, if they are capable of binding to the epitope with very high affinity. Accordingly, the IgG antibody may bind the first epitope of the affinity marker of the invention with an affinity at most 100 x 10 ^'9 mol/1, preferably at most 50 x 10 ^"9 mol/1, more preferably 1O x 10 ^"9 mol/1. still more preferably 5 x 10 ^"9 mol/1, even more preferably 3 x 10 ^'9 mol/1, most preferably 1 x 10 ^"9 mol/1. Additionally, or alternatively, the artificial antibody may bind the second epitope of the affinity marker of the invention with an affinity at most 100 x 10 ^"12 mol/1, preferably at most 50 x 10 ^"12 mol/1, more preferably 10 x 10 ^"12 mol/1, still more preferably 5 x 10 ^"12 mol/1, even more preferably 3 x 10 ^"12 mol/1, most preferably 1 x 10 ^"12 mol/1.

General methods of determining the affinity of an antibody to its epitope are well known to the skilled person and may be adapted to the present epitopes and antibodies.

In one embodiment of the invention the labeled or tagged protein to be purified or used in the methods of the invention may have been or may be produced by a eukaryotic cell, preferably a vertebrate cell, more preferably a mammalian cell, still more preferably a human cell.

Additionally, at least one of the tags may be cleaved off after step b), c) and/or step d) of the methods of the present invention, as appropriate.

Furthermore, the IgG antibody and/or the artificial antibody used in the context of the present invention may be attached to a microarray or chip.

In a preferred embodiment of the invention the first epitope is used for standardization and/or as recovery marker as detailed above in the context of ChIP.

In one embodiment of the invention, the method of purification according to the invention can also be used in order to identify components (prey) which interact with a molecule labeled with the affinity marker (bait) and bind to it. Such bait/prey experiments are familiar to a person skilled in the art.

Yet another subject of the invention relates to the use of the affinity marker of the invention for the purification of a protein and/or for determining or localizing a protein or nucleic acid sequence, particularly from a cell, especially from a eukaryotic cell, preferably from a mammalian cell..

Still another subject of the present invention is a kit comprising

- the nucleic acid and/or the vector of the invention as defined above; and

- optionally an IgG antibody specific for the first epitope as defined above; and

- optionally an artificial antibody specific for the second epitope as defined above.

Additionally, the kit may encompass instructions for carrying out the e.g. the methods of the present invention.

The present invention is additionally described by the following figures and examples, which are not to be interpreted as limiting the scope of protection of the present invention.

FIGURES

Fig. 1 shows a Western blot analysis of MBP from Raji cells of EXAMPLE 2. 50 μg total protein prepared from MBP-transfected or non transfected Rajji cells was separated on an 12% SDS PAGE and transfered onto nitrocellulose. Membrane was probed with anti-MBP antisera (NEB).

Fig. 2 shows a Western blot analysis of MBP-HRPT2 from Raji cells and Jurkat cells of EXAMPLE 2. 50 μg total protein prepared from MBP- HRPT2 transfected or non transfected cells was separated on an 12% SDS PAGE and transfered onto nitrocellulose. Membrane was probed with anti-MBP antisera (NEB).

Fig. 3 and 4 show a Western blot analysis of a MBP IP using mbpoff7 or the unspecific DARP E3 5. Whole cell extracts (50 μg total protein) isolated from Raji cells overexpressing MBP were immunoprecipitated with mbpoff7 or E3 5

as indicated. To monitor MBP anti-MBP antisera was used in the western blot analysis (Fig. 3) Alternatively 20 μl of the samples were analyzed by SDS PAGE and silver staining (Fig. 4).

Fig. 5 shows transcription complexes at TCRβ. Specificity of NC2α Mab in ChIP:

ChIP experiments of NC2alpha I comparison to NC2β, TFIIB and Rpbl and distribution of the complexes over the TCRβ promoter. 20 ng DNA template, resulting from the corresponding ChIP experiments, was analyzed by real-time-PCR using primer pairs representing different TCRβ promoter regions as indicated in the figure (- PMA and + PMA: first and second column of each condition, respectively).

Fig. 6 shows expression and purification of synAB of PrP (A) and GCN4 (B).

Fig. 7 shows pKG2 vector.

Fig. 8 shows the results of a luciferase assay. HeLa cells transfected with pKG2

(stable cell line), induced with Doxcycline (1 μg/ml) for 24 hours.

Fig. 9 shows Western blots of different constructs expressed in various cells.

A: Western blot of MED25 in Raji cells stably transfected with pMH28 (Med25-GCN4-NC2). 15 μg nuclear extract protein from Raji-pMH28 and Raji cells was separated on 12 % SDS-PAGE gel and transferred onto nitrocellulose. Membrane was probed with anti MED25 antibody, second antibody: anti-rat-HRP. The arrow shows the expression of MED25-tagged fusion protein.

B: Western blot of HRPT2 in Raji cells stably transfected with pAK35 (NC2-GCN4-HRPT2). 15 μg nuclear extract protein from Raji-pAK35 and

Raji cells was separated on 12 % SDS-PAGE gel and transferred onto nitrocellulose. Membrane was probed with anti HRPT2 antibody, second

antibody: anti-rabbit-HRP. The arrow shows the expression of HRPT2- tagged fusion protein.

C: Western blot of NC2 from Raji cells stably transfected with pMH28 (NC2-GCN4-MED25). 15 μg nuclear extract protein from Raji-pMH28 and

Raji cells was separated on 12 % SDS-PAGE gel and transferred onto nitrocellulose membrane. Membrane was probed with anti NC2 antibody, second antibody: anti-mouse-HRP. The arrow shows the expression of NC2-tagged fusion protein.

D: Western blot of NC2 in mouse ES cells and human Jurkat cells. Nuclear extract protein from mouse ES cells and human Jurkat cells were separated on 4-12 % SDS-PAGE gel and transferred onto nitrocellulose membrane. Membrane was probed with anti NC2 antibody, second antibody: anti-rat- HRP. The arrow shows the expression of NC2 protein only in human Jurkat cells.

Fig. 10 shows Western blots of different constructs expressed in various cells.

A: ScFv/GCN4 IP; 1 ^st antibody: anti FLAG; 2 ^nd antibody: anti-mouse-HRP

Lane 1 : Epoxybeads-ScFv/GCN4 IP in non crosslinked chromatin from pKG2-HeLa ;

Lane 2: Epoxybeads/GCN4 IP in crosslinked chromatin from pKG2-HeLa;

Lane 3: Epoxybeads-ScFv/GCN4 IP in crosslinked chromatin from pKG2- HeLa.

B: ScFv/GCN4 IP; I ^s1 antibody: anti-HA; 2 ^nd antibody: anti-mouse-HRP Lanel : Epoxybeads-ScFv/GCN4 IP in non crosslinked chromatin from pKG2-HeLa ; Lane 2: Epoxybeads/GCN4 IP in crosslinked chromatin from pKG2-HeLa;

Lane 3: Epoxybeads-ScFv/GCN4 IP in crosslinked chromatin from pKG2- HeLa.

C: ScFv/PrP IP; 1 ^st antibody: anti-NC2; 2 ^nd antibody: anti-rat-HRP Lane 1 : Epoxybeads-ScFv/PrP IP in crosslinked chromatin from pCG26- HeLa; Lane2: Epoxybeads/PrP IP in crosslinked chromatin from pCG26-HeLa.

Fig. 11 shows RT-PCR results of yield DNA purified with sAB bound different supports.

A: RT-PCR result of yield DNA in crosslinked Jurkat cells to synthetic antibody (sAb) on protein L agarose.

B 1 % BSA in PBS,

D 5 X Denhardt's solution,

S salmon sperm DNA (100 μg/ml),

P PEG800 solution (0.5%). 20 μl proteinL agarose was used for coating of 5ug sAb.

Results:

AVERAGE STDEV proL-sAb+BSA 0.04Si S5 0.006245 0.0082 ! 8 0.020883 0.019323 proL-sAb+B+D 0.019029 0.007418 0.010036 0.012161 0.004973 proL-sAb+B+S 0.01 132 0.00901 1 0.009915 0.010082 0.00095 proL-sAb+B+D+S 0.006883 0.007418 0.01099 0.00843 0.001823 proL-sAb+B+D+S+P 0.005458 0.01 1605 0.008532 0.003074

B: RT-PCR result of yield DNA in crosslinked Jurkat cells to synthetic antibody (sAb) on epoxy magnetic beads.

B 1% BSA in PBS,

T yeast tRNA ( 10 μg/ml),

S salmon sperm DNA (100 μg/ml),

P PEG800 solution (0.5%). 10 μl epoxy magnetic beads was used for coating of 10 μg sAb.

Results:

AVERAGE STDEV epoxy-sAb-BSA 0.000738 0.002295 0.00395768 0.00233 0.001315 epoxy-sAb-B+T 0.002447 0.004367 0.003407 0.00096 epoxy-sAb-B+S 0.003251 0.00303894 0.003145 0.000106 epoxy-sAb-B+T+S 0.000837 0.001638 0.001237 0.0004 epoxy-sAb+B+T+S+P 0.002367 0.01980072 0.01 1084 0.008717

C: RT-PCR result of yield DNA in crosslinked Jurkat cells to synthetic antibody (sAb) on Tosylactivated magnetic beads. B 1% BSA in PBS,

T yeast tRNA (10 μg/ml),

S salmon sperm DNA (IOO μg/ml),

P PEG800 solution (0.5%).

10 μl tosylactivated magnetic beads was used for coating of 10 μg sAb. Results:

AVERAGE STDEV tosyl-sAb-BSA 0.003272 0.004128 0.00294486 0.003448 0.000499 tosyl-sAb-B+T 0.00259 0.000975 0.001783 0.000808 tosyl-sAb-B+S 0.004373 0.004373 0 tosyl-sAb-B+T+S 0.003551 0.004948 0.00425 0.000699 tosyl-sAb+B+T+S+P 0.007663 0.00710333 0.007383 0.00028

EXAMPLES

EXAMPLE 1

Heterologous expression and purification of the Designed Ankyrin Repeat Protein mbpofπ (DARP mbpoff?)

The DARP mbpoff7 was expressed and purified as described in Binz et al., 2003, J. MoI. Biol. 332, 489-503. SDS PAGE of the purification of the DARP mbpoff7. Critical steps during the purification of the recombinant protein mbpoff? (as indicated) were analyzed on a 12 % SDS gel following a coomassie blue staining.

EXAMPLE 2

Expression of MBP and MBP-tagged protein in human cells

The MBP sequence was cloned as an N-terminal tag in the expression vector pREP4. For transfection of the construct of about 5 x 10 ⁶ Raji cells were pelleted and washed once with PBS. Cells were resuspended in 400 μl growth medium (RPMI) without serum per sample. The cell suspension was then distributed in a precooled electroporation cuvette (Gene Pulser Cuvette, BioR.ad Cat. N. 165-2088) where it was mixed together with a total amount of 15μg DNA and left on ice for about 10-20 minutes.

For the electroporation the cuvette was connected to a Gene Pulser electroporator (BioRad) and the conditions set to a capacitance of 1000 μF and a voltage of 230 V.

After the electric shock, cells were resuspended in 5 ml medium per sample, transferred in 25 cm ² flasks and incubated at 37°C and 5% CO ₂ . 48 hours later, cells were harvested and pellets were resuspended in 200 μl 1 x GENNT buffer (5% glucose, 5 mM EDTA, 0.2% Nonidet P40, 15OmM NaCl, 50 mM Tris-Hcl pH 8.0, containing protease inhibitors). Samples were incubated for 15 minutes on ice, vortexed and centrifuged for 10 minutes at 13200 r.p.m. at 4°C. The supernatant was transferred in fresh tubes, protein concentration was estimated by Bradford and 50 μg total protein were subjected to western blot analysis.

EXAMPLE 3

Immunoprecipitation of MBP out of Raji cell extracts with or without previous crosslinking of the cells

0.5 x 10 ⁸ stable Raji-MBP cells were harvested for 5 minutes at 1200 r.p.m. and washed once in growth media (RPMI) without serum. The pellet was resuspended in 2.5 ml RPMI media, formaldehyde (37%) was added to a concentration of 1% and cells were shaken on a roller for 9 minutes at room temperature. The reaction was stopped by adding 125 mM glycine and incubation on ice for 10 minutes. Pellets were washed three times in ice cold PBS, resuspended in 5 ml 1 x RIPA buffer (1% NP40, 0.5% deochycolate, 0,1% SDS in PBS, containing protease inhibitors), incubated 10 minutes on ice and sonicated three times for 15 seconds at 20% output setting on an ice/ethanol water bath using a Branson 250 sonifier. The suspension was divided in 2 ml tubes and centrifuged for 15 minutes at 13200 r.p.m at 4°C. Supernatants were transferred in fresh tubes and protein concentration was estimated by Bradford assay.

For extracts from non crosslinked cells the same amount of cells were instead of crosslinking kept on ice and proceeded than the same way as the crosslinked cells.

For preparation of the mbpoff7/E3_5 beads 40 μl Affigel 15 (BioRad) beads were used per reaction. Beads were pretreated following the instructions of the manufactor and incubated over night with 10 μg mbpoff7 per reaction in a total volume of 500 μl TBS ₅₀₀ (50 mM Tris-HCl, pH 8.0, 500 mM NaCl) on a roller at 4°C. Beads were blocked by adding 0.1 ml 1 M glycine ethylester per ml gel and incubation for 1 h at 4°C on a roller. Beads were washed with 5 ml ice cold H ₂ O and equilibrated with 0.5 x RIPA buffer. The equilibrated beads were incubated with 125 μg protein extract and incubated over night at 4°C on a roller. Beads were washed 3 times with 1 ml of washing buffer (TBS-T; 1 x RIPA buffer; 25OmM LiCL 1% NP40, 1% deoxycholate (a); 50OmM LiCl, 1% NP40, 1% deoxycholate (b) or 50OmM LiCl, 1% NP40, 1% deoxycholate, 0.1% SDS (c) as indicated. Finally, beads were resuspended in 30 μl 2 x protein loading dye, incubated for 5 minutes at 95°C and 15μl supernatant were subjected to western blot analysis.

EXAMPLE 4

NC2 ChIP experiments

NC2 ChIP experiments were carried out according to the following protocol adapted from 5 Odom et al., 2004, Science 303, 1378-1381.

(See also http://web.wi.mit.edu/young/pancregulators/).

1. Induction of cells

- Induce cells with 4-OHT (final concentration 1 μM). I O

2. Cell Cross-Linking

- Use 1x10 to 1x10 cells. Split cells the day before harvest. Harvest cells at appropriate cell density (e.g. 5Xl O ³ cells/ml for Jurkat).

- Pool cells, take small aliquot (≤ 100 μl) into Eppendorf cup and resuspend w/ P200 15 to obtain single cells for counting. Determine cell density and total cell number in

Neubauer counting chamber (cell number in 16 small squares x 10 ⁴ = cell number/ml).

- Spin cells down in 250 ml Corning tubes, 1200 rpm, 5 min, RT.

- P-cmove supematent, resuspend cell pellets in ~ 40 ml pre-warmed medium (w/ 10% 0 serum) and pool into 50 ml Falcon tube.

- Spin down, 1200 rpm, 5 min, RT.

- Remove supernatent, resuspend pellet in 43.78 ml medium w/o serum (RT)

Add 37% formaldehyde 1.22 ml 37% CH ₂ O -> 1 % final cone. (Roth, p.a., #4979.1), total volume 45.00 ml. 5 - Incubate on roller table for 9 min at RT, constant agitation.

- Add 2 M glycine 3.00 ml 2 M glycine -> 125 mM (filter sterilized) final cone, total volume 48.00 ml.

- Mix quickly, put on ice for 10 min (suspension will turn yellow upon addition of glycine). 0 - Work on ice from here on.

Spin down, 1200 rpm, 5 min, 4°C.

- Remove supernatent. wash pellet 3 x 50 ml ice-cold PBS.

- Last wash: transfer pellet to 15 ml Falcon tube, spin, remove PBS as much as possible, freeze pellet on dry ice and store at -80°C. Alternatively, continue after last wash directly with step 0.

3. Cell Lysis and Sonication

Add Complete (Roche, # 1 1697498001) protease inhibitor mix to all lysis buffers before use.

- 25 x Complete: Grind 1 tablet in between 2 sheets of balance paper, dissolve fine powder in 2 ml of H ₂ O, aliquot and store (labeled 25xC) at -20°C. - Lysis Buffer 1 (LBl) (final cone): 50 mM Hepes-KOH (pH 7.5), 140 mM NaCl, 1 mM EDTA (pH 8.0), 10 % glycerol, 0.5 % NP-40, 0.25 % 10% Triton X-100

Lysis Buffer 2 (LB2) (final cone): 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM Tris-HCl (pH 8)

Lysis Buffer 3 (LB3) (final cone): 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM Tris-HCl (pH 8), 0.5 % N-lauroyl sarcosine before use supplement with (final cone):

BEFORE sonication: 0.1 % Na-deoxycholate (freshly prepared) and 1 x complete

AFTER sonication: 0.5 % Triton X-100 and 10% glycerol

When using frozen pellets: thaw on ice; when using fresh pellets: add directly 10 ml of Lysis Buffer 1 (LBl) per pellet. Mark pellet volume on tube to check for efficient nuclei prep in step 0.

Rock at 4°C for 10 min. Then spin at 4000 rpm, 10 min, at 4°C (e.g. in Heraeus

Multifuge). Pellet should be smaller (check marks on tube) and look brighter. If this does not happen, repeat step 0. Eventually check nuclei in microscope. - Resuspend each tube of pellets in 10 ml Lysis Buffer 2 (LB2). Rock gently at RT for

10 min. Then spin at 4000 rpm, 10 min, at 4°C.

- Resuspend pellet in 3 ml buffer Ix Lysis Buffer 3 (I x LB3) without Triton X-100 and glycerol. Sonicate the suspension in 15 ml conical tube with a microtip attached to Branson 250 sonifier on ice/ethanol water bath: - Sonifier settings:

Slow mode: 40% output setting, time 30 sec, Pulse On 0,9 sec, Pulse Off 0,1 sec

Sonicate 13 cycles, i.e. 6 min 30 sec in total, allowing the suspension to cool on ice for > 1 min between pulses.

After sonication, add 1/20 volume of 10% Triton X-100 to 0,5% final concentration. Transfer to 1.5 ml tubes and spin out debris at 4°C, 15 min, 13 k rpm in cooled tabletop centrifuge.

- Transfer supernatant (= soluble chromatin) to fresh 15 ml conical tube. Determine "dsDNA" cone, of sample (> 1 : 100 dilution for measurement). Dilute chromatin solution to 1 mg/ml (if possible to 2 mg/ml) "dsDNA" Cone, with LB3 and add glycerol to 10% final cone. Transfer 1 ml aliquots to 1 ,5 ml tubes, set 10 μl aliquot aside for analysis, and snap-freeze 1 ml aliquots in liquid nitrogen.

Store at -80°C.

- Analyze 10 μl sample from 2.6 for chromatin size: add 30 μl (3 volumes) of ChIP EB (see 3.x) to 10 μl sample. Add 2 μl of proteinase K (PCR grade; Roche) and incubate > 2 hours at 65°C to revert crosslinks and digest proteins. Purify DNA w/ Qiaquick PCR purification kit (column format). Load 1 to 2 μg purified DNA onto

1% agarose gel and check for size distribution of fragments.

- If necessary (too large bulk size of fragments), a second sonication might be performed. Settings for this second sonication have to be determined empirically. Be aware that samples now contain 0.5% Triton X-100 from step 2.5. making the chromatin solution more prone to foaming during the second sonication.

4. Immunoprecipitation

- Mix Prot.A/G-Sepharose (FF, GE Healthcare) in a ration of 1 :2 due to different binding capacities. - Wash beads 2-3x with icecold I xPBS in lubricated tubes (centrifuge ≤ 3k rpm at 4°C for l '). Block beads by incubation with 1 xPBS / 0,5% for 30' at 4 ⁰ C rocking.

- Thaw sufficient aliquots of X-linked chromatin samples. Per ChIP calculate to use 100 μl of 2 mg/ml or 200 μl of 1 mg/ml X-linked chromatin extract. Thus, an aliquot of 1 ml is sufficient for 5 ChIPs (if 1 mg/ml cone.) or 10 ChIPs (if 2 mg/ml cone).

Pre-clear chromatin extract by incubating with blocked beads: use 10 μl (dbv) of appropriate beads per 100 μl of chromatin extract. Thus, a total of 100 μl (dbv) of

blocked beads is used per 1 ml aliquot of chromatin extract. Add 200 μl of 50% blocked beads slurry (in 1 x LB3) per 1 ml aliquot. Incubate with constant agitation at 4°C for 30-60 min (lubricated tubes).

- In parallel, pre-coat blocked beads with appropriate antibodies. Per ChIP typically 2- 10 μg antibody is used; if working with hybridoma tissue culture sups, use 100-200 μl of sup instead. Use 15-20 μl (dbv) of appropriate beads per ChIP.

- Assemble precoating mix (in lubricated tubes): 40 μl 50% blocked beads PBS slurry(= 20 μl dbv blocked beads), xjαl antibody (= 1 μg), add 400 μl w/ cold PBS

- Incubate with constant agitation at 4°C for 30-60 min. - Spin down 1 min at 4°C, 3000 rpm, and remove sup. Wash pellet Ix w/ 1 ml cold IxPBS. Spin down, remove sup. Resuspended pellet in 1 volume Ix LB3 (0.5% Triton X-100, Ix Complete, but w/o glycerol).

- Assemble ChIP sample (in lubricated tube): 40 μl antibody-coated beads (50% slurry in Ix LB3), 100 μl (or 200 μl) of pre-cleared chromatin extract (from 0) - Incubate ChIP samples for > 2 hours (typically o/n) with constant agitation at 4°C.

5. Washing, eluting, and reverse cross-linking

Spin beads, 3000 rpm, 30 sec, 4°C

- Wash beads 6x with imi Ix RIPA-Buffer - Wash buffer (2x RIPA buffer) (final cone, if Ix): 50 mM Hepes (pH 7.6), ImM

EDTA, 1 % NP-40 (IPGEL), 0.5 M LiCl

Add to freshly prepared Ix RIPA-buffer: 0,1 % Na-deoxycholate (freshly prepared),

Ix Complete

Wash beads Ix with 1 ml TE-buffer + 5OmM NaCl - Add 100 μl prewarmed (65°C) Elution buffer and rock samples for 10 min at

1400 rpm and 65°C in thermomixer.

Elution buffer: 5OmM Tris pH 8.0, 10 mM EDTA, 1% SDS

Spin down beads, 13k rpm, 1 min, RT; transfer all of the 100 μl sup in 1.5 ml non- lubricated tubes. - Fill Inputs ad 100 μl with H ₂ O and add 3x volumes of Elution buffer.

Reverse x-link O/N 65°C.

6. RNase A, Proteinase K

- Add Ix volume of TE-buffer and then add RnaseA (lmg/ml) so final is -0.05 μg/μL: 5μl per ChIP and lOμl per Input. Incubate for 1-2 h at 37°C. - Add proteinase K so final is 0.2 μg/μL (-2.5 μL/250 μL rxn): 4 μl per ChIP and 8 μl per Input.

Incubate for 2 h at 56°C. Store at -20°C. Alternatively, continue directly with step 0.

7. DNA-purification

Add once Ix vol of PCI-mix (25:24:1, Fa. Roth) and vortex 20-30 sec.

- Spin samples, 13k rpm, RT, 5 min and transfer aqeuos (upper) phase in fresh 1,5 ml tube.

- Prepare mastermix for EtOH-precipitation: l/25x vol 5 M NaCl, 1 ,5 μl Glykogen, 2x vol 100% EtOH, icecold, add mastermix to samples, mix and store samples at -80°C

(> 30 min) or -20°C (> 2 h).

- Spin samples, 13k rpm, RT, 5 min and discard supernatant. Wash pellet with 500 μl ice-cold 70% EtOH and spin again 13k rpm, RT, 5 min.

Dry peiiet, then resuspend in 50 μl 10 rriM Tris/HCi. Rock samples for 15 rnin at RX in the thermomixer.

- Determine "dsDNA" cone, of sample (> 1 : 100 dilution for measurement). Load 0.5 to 2 μg purified DNA onto 1% agarose gel and check for size distribution of fragments.

8. ChIP-analvsis

Use -20 ng as template in the PCR-rxn.

EXAMPLE 5

The expression and purification of the synthetic antibody (synAB) against GCN4 and BoPrP

The synABs were developed by Prof. Plueckthun (University of Zurich). One 30 KD

synAB is against a 33 amino acid GCN4-peptide from yeast, the other 30 KD synAB is against 15 peptides Bovine Priontag. Both of them have picomolar affinity to the target- peptide, which is expressed in E. coli with His-tag and FLAG-tag (FLAG-PrP tag, Peptide sequence: DYKDDDDK-GAG-DYKDDDK-GS-GQGGGTHGQWNKPSKP; SEQ ID NO: 10). Expression and purification of synAB of PrP and GCN4 is shown in figure 6 A and B. respectively.

Single chain antibody expression protocol

1. Transformation of pAK400 vector to competent cell (SB536) Ice 30 min, 42 °C, 45 sec, ice lmin

Add 1 ml LB medium, 37 °C for 1 hour

Take 100 μl and plate on agar-plate (chloramphenicol), 37 °C overnight.

2. Pick one clone and put it into 50 ml LB medium (30 μg/ml chloramphenicol), 37 °C shake at 200 rpm, overnight 3. Take 20 ml and put them into 500 ml LB medium, 37°C, shake at 200 rpm, until

OD=0.5 4. Add IPTG, final concentration 1 itiM, at 25°C shaking incubation for 4 hours

Centrifuge at 8000 rpm for 10 min, 4°C, resuspend the pellet with 20 ml PBS, then add lysozyme 5. Sonicate at 40% output, 15 sec pulse on, 45 sec pulse off, together 6 min, 24 times,

15sec. After sonication, centrifuge at 20,000 rpm for 20 min, 4°C

ScFv purification protocol

1. Wash the column with 20 ml H ₂ O and then with 20ml PBS, cool it. 2. Add 4 ml Ni-NTA (50 %), equilibrate with 20 ml PBS

3. Load the protein onto the column

4. Wash the column with 20 ml washing buffer (20 mM imidazol-PBS)

5. Elute the column with elution buffer (250 mM imidazol-PBS), collect the elute flux in 500 μl for 15 tubes. 6. Wash the column with water and PBS, store at 4°C

Electrophoresis and Protein dialyze

Separate the protein on 4-12% SDS-PAGE gel. Transfer the protein sample into the dialyze bag. in dialyzing buffer (PBS) stir overnight at 4 degree.

EXAMPLE 6

Expression of GCN4 or prion-tagged interested protein

GCN4 or prion-tagged interested proteins (vector construction)

We constructed an EBV based tet-on vector with GCN4 tag (see figure 7). Then we transfected the vector into HeLa cells and produced a stable cell line.

Transection of cells with the construction and generation of stable cell lines

The day before transfection, seed 6 x 10 ⁶ HeLa cells on 100 mm ² cell culture plate, dilute 6 μg DNA (pKG2 or H ₂ O) dissolved in 50 μl TE and 100 μl DMEM (no serum and antibiotics), mix and add 50ul polyFect (Invitrogen) reagent, mix and incubate for 10 min at room temperature (RT), wash the cells with 8 ml PBS, add 7 ml fresh DMEM (with serum and antibiotics), add 1 ml complete medium to reaction tube, mix and add to the plate, incubate the cells at 37°C plus 5% CO ₂ overnight, add puromycin for selection (initial concentration 3 μg/ml), when the control cells died, stop the selection and add puromycin (concentration 0.5 μg/ml) to keep cell growth.

Detection of tagged proteins

Nuclear extracts were prepared according to the following protocol:

1. Centrifuge the cells at 1000 rpm for 7 min at 4 ⁰ C, wash the pellet with cold PBS 5 ml, resuspend he pellets in 300 μl NEXA solution (up to 10 ⁷ cells), incubate on ice for

1 Omin, add 10% NP-40 10 μl for 5 min on ice, transfer the extract in mortar and grind on ice, transfer the whole solution into a new EP tube, spin down at 1000 g for 5 min, take the supernatant (cytoplasm) and freeze in liquid nitrogen and save at -80 °C

2. Add NEX B buffer 60μl (=270/130 x pellet Volume μl) to the pellet incubate at 4 °C on a roller for 30 min, spin down at maxi speed for 10 min, take the supernatant and freeze in liquid nitrogen, save at -80 °C (nucleic extraction)

Western Blot

Western blots were prepared according to the following protocol:

1. Separate the protein on SDS-PAGE Mini-gel (4- 12%)

2. Transfer to nitrocellular membrane at 15volt for 45min 3. Ponceau S staining 2 min and wash away the staining

4. Wash the membrane again till the red staining is away and block the membrane for 1 hour in TBS-T containing 5% milk powder at RT

5. Add 1 ^st antibody diluted in TBS-T (1% milk powder, 0.02 % Tween 20) and incubate in cold room overnight, 3 X 15 min wash in TBS-T at room temperature. 6. Add 2 ^nd antibody, for 30min in TBS-T (1% milk powder, 0.02 % Tween 20), 3 X 15 min wash in TBS-T at room temperature

7. Wash the membrane with TBS and then water and then ECL reaction: black and white bottle solution 1 ml + 1 ml 1 : 1 mix and put onto the membrane for 2 min, then suck away the solution, and exposure in the dark room.

The results are shown in figures 9 and 10.

Luciferase Assay

The luciferase assay was carried out according to the following protocol: Plate the stable cell line of HeLa-pKG2 cells on 12-well plate (2 x 10 ⁵ cells/well) overnight, add doxycycline (1 μg/ml) for 24 hours, then wash the cells with PBS twice, add 150 μl Ix lysis buffer (Promega), vortex for 10 second, incubate 10 min at RT, transfer the lysis to new eppendorf (EP) tube, spin down at 13,000 rpm for 15 min at 4 °C, pipette

50 μl to 96-well plate for measure.

The results of this experiment are shown in Fig. 8.

EXAMPLE 7

Immunoprecipitation of pKG2-HeLa cells extracts with or without previous crosslinking of the cells

The experiments were carried out according to the following protocol:

Preparation of the crosslink and none crosslink chromatin from pKG2-HeLa cells Take 10 cells, centrifuge at 12krpm for 5 min Separate the pellet into 2 falcon tubes - Pellet 1 was kept on ice (no crosslink)

Pellet 2 will be crosslinked

- Pellet 2: add 2.5 ml DMEM (no serum) + 67 μl 37 % formaldehyde, incubate at RT for 9min,then add 156 μl of 2M glycin and incubate on ice for lOmin

- Wash pellet 1 and pellet 2 with 15ml cold PBS 3 times. - Then resuspended in 5 ml RIPA buffer + PI (protein inhibitor), and incubate on ice for 10 min

Then sonicate at 20 % input, pulse on 15sec, pulse off 45sec, 3 times

- Centrifuge at 13.2krpm for 15min at 4 ⁰ C

Collect the supernatant and measure the concentration by Bradford assay.

IP protocol

- 50 μl Epoxy-ScFv(GCN4) beads + 250 μg pKG2-HeLa (crosslink or none crosslink chromatin)

50 μl Epoxy beads+250 μg pKG2-HeLa (crosslink or none crosslink chromatin), as control

25 μl beads for each IP sample

Incubate the beads with lysate at 4 °C for 4 hours

- Wash the beads with TBS-T 3 times

Then add 30 μl 2X loading dye and boil at 95 °C for 5 min - Spin down at maxi speed and collect the supernatant

Analysis on SDS-PAGE gel, continue with Western blot as detailed above.

The results are shown in figure 10.