EP1513935A1 - Vecteurs destines a l'expression de proteines biotinylees dans des cellules mammaliennes et leur utilisation pour identifier les interactions proteines - acides nucleiques in vivo - Google Patents

Vecteurs destines a l'expression de proteines biotinylees dans des cellules mammaliennes et leur utilisation pour identifier les interactions proteines - acides nucleiques in vivo

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Publication number
EP1513935A1
EP1513935A1 EP03756089A EP03756089A EP1513935A1 EP 1513935 A1 EP1513935 A1 EP 1513935A1 EP 03756089 A EP03756089 A EP 03756089A EP 03756089 A EP03756089 A EP 03756089A EP 1513935 A1 EP1513935 A1 EP 1513935A1
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Prior art keywords
protein
interaction
interest
dna
vivo
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EP03756089A
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German (de)
English (en)
Inventor
Vasily Ogryzko
Heike Lehrman
Cristele Gilbert
Antoine Viens
Undine Mechold
Annick Harel-Bellan
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Centre National de la Recherche Scientifique CNRS
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Centre National de la Recherche Scientifique CNRS
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the invention relates namely to a biotinylation process, to vectors for expression of biotinylated proteins in mammalian cells in vivo, and their use for identification of protein-protein and protein-nucleic acid interactions in vivo.
  • first method to study protein-protein interaction is to purify the native complexes of the proteins directly from the cells and identify their components.
  • the methods of biochemical separation, such as chromatography, centrifugation, etc. could be used for this purpose, provided that the presence of the complex in the fractions could be detected in some assay. If the function of the complex is known, such an assay could be based on its enzymatic activity, or on the ability to bind a particular ligand.
  • the purification scheme could include, as a crucial step, affinity chromatography with antibodies (some time obtained serendipitously, as in the case of autoantigen antibodies) against one of the components of the complex.
  • the affinity chromatography could be performed with the ligand, instead of the antibodies.
  • the identification of the components of the complex can be accomplished by a number of methods developed in the field of proteomics, such as mass-spectrometry or Edman-degradation sequencing.
  • the two-hybrid system (Legrain P and al, EERS Xett 480, 32-6 (2000) ; Tucker CL and al ,.Trends Cell Biol 11, 102-6 (2001) ; Serebriiskii IG, and al, .Biotechniques 30, 634-6 (2001)) .
  • This approach allows for in vivo detection of the protein-protein interactions by fusing two putative partner proteins to two functional domains of a particular signal transducing protein (STP), most commonly a transcription factor, whose activity is easily detectable in a particular experimental setup. For the signal transduction to occur, these two STP domains have to be in a close physical proximity, which will be provided in the case of the interaction between the protein partners.
  • STP signal transducing protein
  • each ORF is fused with an STP domain.
  • This library is then transformed into the cells that express the protein of interest fused with the second STP domain.
  • the clones that express the putative interaction partners of the protein of interest are selected based on the functional assay for the particular STP.
  • the affinity chromatography with immobilized protein of interest In this setup, the protein of interest is immobilized on a solid chromatographic support, either by direct chemical crosslinking, or via a fusion with such widely used interacting protein domains as glutathione S-transferase (GST), His etc.
  • GST glutathione S-transferase
  • the immobilised protein is incubated with a protein mixture (tissue, cellular, nuclear extract or others). After extensive washing, the retained polypeptides are identified by a number of methods developed in the field of proteomics, such as mass- spectrometry or Edman-degradation sequencing.
  • Second limitation of both approaches is that they are designed to pick only potential interaction partners from the pool of presented candidates. Whether these potential partners reflect the actual interactions that occur in the cell has to be confirmed by in vivo analysis of the homologous (e.g. mammalian) cell system. Usually it is achieved by coimmunoprecipitation method, which requires antibody development, with all problems the accompanying this work.
  • Protein-DNA/RNA interactions For the search of novel protein-DNA/RNA interactions, the approaches, adaptable for a high throughput analysis, can be broadly divided into two classes: in vivo and in vitro approaches. 1.
  • Generic in vivo approach based on transformation of cells with a library of sequences in a setup that allows to select cells according to a particular phenotype, that depends on the interaction between probed DNA and protein (Sieweke M. Methods MolBiol 130, 59-77 (2000)).
  • Generic in vitro approach affinity chromatography
  • the limitations here are very similar to the previous case: 1. Both approaches are designed to detect mostly binary interactions (in particular, they do not take into account chromatin context of the DNA). 2. They can find only potential binding sites.
  • Chromatin ImmunoPrecipitation (ChlP) (Kuo MH and al., Methods 19, 425-33. (1999) ; Orlando V., Trends Biochem Sci. 25, 99-104. (2000)).
  • ChlP Chromatin ImmunoPrecipitation
  • X-ChlP the DNA is crosslinked in vivo to bound proteins, then sheared and the nucleoprotein complexes are immunoprecipitated with antibodies directed against the studied proteins. If the putative DNA target of the protein is known, the interaction can be detected by PCR. Lately, ChlP was used to identify unknown in vivo targets (Ren B, et all Science 290, 2306-9 (2000)).
  • ChlP ChlP assay
  • Epitope tagging is known in the prior art as a technique comprising protein modification (usually by recombinant DNA technology) that adds to the native protein a short peptide sequence (epitope tag), recognizable by a specific antibody (Fritze CE and al., Methods Enzymol 327, 3-16 (2000)).
  • the epitope tag can be put in any place of the protein, usually on its N- or C-terminus.
  • recombinant cell lines are constructed that express the epitope tagged protein. The protein of interest can be then detected and purified with the antibody directed against the epitope tag.
  • epitope-tag One limitation of the known epitope-tag system is a possibility of the epitope-tag to affect the function and interaction characteristics of the protein of interest. So far, epitope-tagging systems used in mammalian cells for detection, localization and purification of proteins such as Flag-, HA-, myc-tags (Ikura and al. Cell 102, 463-73 (2000) ; Rowan AJ and al, Hum Mutat 9, 172-6 (1997)) are not enough efficient due to the insufficient interaction of the epitope tag and antibody. As a result, usually high nonspecific background is observed during purification of complexes.
  • biotin as epitope.
  • the biotin- avidin interaction is one of the strongest and most specific interactions used in molecular biology (Wilchek M and al, Trends Biochem Sci 14, 408-12 (1989)). Its dissociation constant is 10 " M, which is several orders of magnitude stronger than for most of antibodies.
  • the inventors have used a protein biotinylation technique.
  • the inventors have used the biotin acceptor domains for the identification of the protein-protein or protein- nucleic acid interactions in vivo, by copurifying the complexes of the biotinylated proteins of interest with the interaction partners (proteins or DNA/RNA) and identification of these partners.
  • Such engineering of proteins to target them for biotinylation can be considered a version of epitope-tagging, as it shares most of its features, except that the tag is recognized not by an antibody but a (strept)avidin moiety.
  • Very few proteins in the cell are biotinylated naturally, therefore, purification and detection of a given biotinylated protein should be very specific as well.
  • the proteins fused to the biotin acceptor domains are already biotinylated in vivo, due to the endogenous BirA enzyme activity. If this activity is limiting (in the case of overproduction of the protein in the host cells, or use of heterologous system) BirA can be coexpressed in the cells together with the protein of interest, which allows to achieve high levels of biotinylation in vivo.
  • free biotin vitamin H
  • biotinylation tag compared to other tags for the particular task of the complex purification, is based on the strength and specificity of the biotin- avidin interaction.
  • Other references which are related to the subject matter of the present invention are the American patent published under number US 5,252,466 ("Fusion proteins having a site for in vivo post-translation modification and methods of making and purifying them"), the international patent application published under number WO 95/04069 ("Biotinylation of proteins”), the publication Sibler AP and all (“In vivo biotinylated recombinant antibodies: high efficiency of labelling and application to the cloning of active anti-human IgGl Fab fragments", J Immunol Methods, 1999 Apr 22;224(1- 2): 129-40), the publication Duffy S and all ("Site-specific, enzymatic biotinylation of recombinant proteins in Spodoptera frugiperda cells using biotin acceptor peptides", Anal Biochem.
  • the inventors had to solve not only the problem of using a short tag that does not disrupt the function of the protein of interest, but also to obtain an appropriated coexpression of the biotinylation enzyme (for example, BirA) and of the protein of interest in the same cells.
  • the biotinylation enzyme for example, BirA
  • One potential problem with the BirA expression is the large volume of the mammalian cells compared to the bacterial cells, which would negatively affect the efficiency of biotinylation.
  • the amounts of BirA required to achieve efficient in vivo biotinylation might negatively interfere with the cell functioning, for example, due to undesirable biotinylation of other proteins in the cell. This will also lead to higher nonspecific background during purification.
  • the inventors have achieved the 100 % biotinylation efficiency, at the same time keeping the levels of the enzyme on the minimal necessary level.
  • the inventors have succeeded to get efficient biotinylation, using physiologically (low) biotin concentration in the mammalian cell growth medium, less than 1 mg/L
  • the invention relates according to a first aspect to a method for expressing in eukaryote cells a modified target protein of interest comprising expressing (i) a protein of interest and (ii) a modifying enzyme that achieves the modification of said target protein, wherein the target protein of interest and the modifying enzyme are coexpressed from the same RNA.
  • the vector used for the expression of the modifying enzyme from the same message RNA as the target protein comprises an Internal Ribosome Entry Site (IRES).
  • the modifying enzyme is a biotinylating enzyme that achieves biotinylation of the protein of interest, and the target protein is fused to at least one biotin acceptor domain (BAD).
  • BAD biotin acceptor domain
  • the vector used for efficient biotinylation of the protein of interest in vivo in mammalian cells preferably contains as essential part, BAD domain and BirA, driven by IRES.
  • the invention provides a method to express in vivo in mammalian cells biotinylated versions of the proteins of interest.
  • the inventors have used a set of vectors that express the protein of interest fused with a short biotin acceptor domain. These vectors also express biotinylation enzyme BirA, required for biotinylation of this domain in vivo.
  • the IRES are known from the one skilled in the art (1988, Pettetier and Sonenberg, Nature, 334:320-325) : particular virus mRNAs containing specific sequences internal to the mRNA are translated by an unusual mechanism of "internal ribosome binding" that enable a ribosome to bind and initiate translation. The sequences are termed "Internal Ribosome Entry Site” (IRES).
  • IRES Internal Ribosome Entry Site
  • the eukaryotic ribosomes recognize IRES.
  • the IRES used in the present invention can be derived from either a viral or cellular gene that undergoes cap-independent translation.
  • the IRES is derived from a picomavirus, such as polio virus or encephalomyocarditis virus.
  • the biotinylated protein of interest is expressed via transient transfection.
  • the biotinylated protein of interest is expressed via generation of stably expressing cell lines.
  • the biotinylated protein of interest is typically expressed from a vector allowing an inducible expression.
  • the transfected cells are from a tissue sample, and the tissue sample is mammalian, preferably human (eventually biopsied sample).
  • the invention relates to a method for intracellular localization of the biotinylated protein.
  • the localization is determined using incubation of cells with streptavidin avidin conjugated to a detectable marker (fluorescent, radionucleide etc ... ) and observed in a microscope.
  • the localization is determined via incubation of the sample with streptavidin/avidin coated gold beads and observed in electron microscope.
  • the invention relates to a method for in vivo labeling of biotinylated protein typically by incubating the cells expressing the biotinylated protein in the presence of radioactive biotin.
  • the labeled protein is detected in fractions after biochemical separation by autoradiography.
  • biotinylated protein is detected in fractions after biochemical separation via its interaction with avidin (streptavidin).
  • the detection is based on western blot analysis.
  • the biotinylated protein is purified from the cells.
  • biotinylated protein is purified by affinity chromatography with the immobilized streptavidin, avidin, its derivatives (i.e. monomeric avidin) or antibiotin antibodies.
  • avidin derivative is monomeric avidin.
  • the purification of the biotinylated protein may comprise an additional affinity chromatography step.
  • the additional chromatography step is for instance 6xHis tag.
  • the biotinylated protein is purified in a complex with interaction partners of the protein, typically other polypeptides and nucleic acids.
  • biotinylated protein is purified in mild conditions.
  • the cells are treated with crosslinking reagents and the said complex is purified in stringent conditions.
  • the results are compared with a positive control, typically a known protein and a negative control, which correspond to cells that do not express the biotinylated protein (mock purification).
  • the complex can be further analyzed with biochemical, mass-spectrometrical and other methods.
  • this further analysis of the interaction partners is performed directly on a chromatographic support, without further elution of the protein of interest or/and the interaction partners.
  • the further analysis is performed after removal of the chromatographic support ; for instance the epitope-tag includes a protease cleavage site and the chromatographic support is removed by a protease cleavage.
  • the complex can also be assayed on enzymatic activity, such as histone acetyltransferase, kinase, methyltransferase.
  • the effect of the interaction partners on the enzymatic activity of the protein of interest is assayed with a high precision compared to the prior art methods..
  • the complex can be also analyzed on the presence of interaction partners.
  • the pattern of protein interaction partners is visualized via one or two dimensional polyacrilamide gel separation.
  • the gel is stained with silver or coomassie or fluorescent stains such as Sypro Ruby, to detect the protein interaction partners.
  • the protein interaction partners are chemically biotinylated before separation and visualized on a western blot with streptavidin, avidin and other biotin-binding moieties conjugated with a detectable label (such as peroxidase, phosphatase, FITC for fluorescein isothiocyanate etc).
  • the protein interaction partners of the in vivo biotinylated protein are identified by mass- spectrometry.
  • Said mass-spectrometrical identification can be performed after gel separation of the interaction partners, or after direct protease digestion of the complex, separation of the complex mixture of peptides with one or two-dimensional mass-spectrometry and identification of the peptides with mass-spectrometer. Said digestion is performed directly on the chromatographic support, without prior elution of material, or after elution of the interaction partners in conditions that disrupt the interaction of the said partners with the biotinylated protein.
  • said digestion is performed after elution of the interaction partners in conditions that disrupt the interaction of the biotinylated partner with chromatographic support.
  • the said conditions are 8 M Guanidine HC1 with pH 1.5.
  • the epitope tag includes a protease cleavage site and the complex is released after protease digestion.
  • the said protease is TEV (Tobacco Etch Virus).
  • DNA/RNA is monitored by purification of the said protein complexed with
  • the protein is typically crosslinked in vivo to DNA RNA via chemical or other crosslinking method.
  • the crosslinking reagent is formaldehyde.
  • the presence of DNA/RNA in the immunoprecipitation is detected by PCR.
  • RNA is converted to cDNA by reverse transcription before PCR amplification.
  • the said DNA RNA in the immunoprecipitation is directly labeled without prior amplification with PCR.
  • the DNA/cDNA obtained after biotin affinity purification is cloned.
  • DNA is cloned directly or after PCR amplification.
  • the invention relates to a nucleic construct comprising a
  • DNA sequence encoding a protein of interest sequence an epitope tag, a DNA sequence coding for a modifying enzyme, an IRES sequence.
  • the nucleic construct comprises a DNA sequence encoding a protein of interest, a BAD domain, a BirA sequence, an IRES sequence capable of conducting the coexpression from the same RNA of the protein of interest fused with the BAD domain, and of the BirA.
  • the invention also relates to a vector for efficient modification, namely for efficient biotinylation, of a protein of interest in vivo in mammalian cells, comprising such a nucleic construct.
  • the invention also relates to an eukaryote host cell comprising said vector, preferably mammalian host cells, most preferably, human host cells.
  • Suitable host cells include human fibroblast cell lines (e.g., HeLa cells, 293T cells, 293 cells, W238 cells and HOS cells), human T-cells (e.g., Jurkat, CEM and H9 cells), monkey cell lines (e.g., CVl cells and COS cells), rodent cell lines (e.g., 3T3 cells, L cells, C127 cells and BHK cells).
  • the constructs can be introduced using any of a variety of techniques and agents, including electroporation, calcium phosphate precipitation, DEAE-dextran, lipofectin, cationic lipids and viral (e.g., retroviral) infection.
  • the invention also relates to a kit (A) for transient expression of protein of interest in vivo, comprising:
  • the invention also relates to a kit (B) for generation of stable cell lines expressing the protein of interest in vivo, comprising:
  • kits for in vivo localization of the protein of interest comprising :
  • the invention also relates to a kit for visualization of the protein interaction partners of the protein of interest, comprising : • A kit (A) or (B),
  • biotin • Activated biotin. • Streptavidin or avidin or other biotin-interacting moieties conjugated with a detectable label, for detection of the biotin-labeled interaction partners,
  • kits for identification of the protein interaction partners of the protein of interest comprising : • a kit (A) or (B),
  • the interaction partners are identified by mass-spectrometry.
  • the invention also relates to a kit for detection of the DNA-protein interaction in vivo comprising :
  • the invention also relates to a kit for identification of DNA (RNA) targets in vivo comprisin :
  • the invention also relates to a method for screening a test compound for its ability to modulate interaction between a protein and a protein interaction partner comprising : i) preparing a nucleic construct described above, ii) introducing said construct into a vertebrate host cell and culturing said host cell in the presence and absence of said test compound, and iii) measuring the level of interaction of the interaction partner with the biotinylated protein of interest in the presence and absence of said test compound, a difference in the level of expression of said indicator gene in the presence of said test compound, as compared to the level of biotinylation in the absence of said test compound, being indicative of a test compound that modulates interaction of said protein with said protein interaction partner.
  • the invention relates to a method for screening a test compound for its ability to modulate in vivo interaction between a protein and a protein or a nucleic acid interaction partner comprising : i) preparing a construct according to claim 41 or 42 ii) introducing said construct into a vertebrate host cell and culturing said host cell in the presence and absence of said test compound, and iii) measuring the level of interaction of the protein or the nucleic acid of interest with the biotinylated protein in the presence and absence of said test compound, a difference in the level of interaction of said protein in the presence of said test compound, as compared to the level of interaction in the absence of said test compound, being indicative of a test compound that modulates interaction of said protein with said protein or nucleic acid interaction partner.
  • antibody refers to antibodies and antibody fragments that retain the ability to bind the epitope that the intact antibody binds, whether the antibody or fragment is produced by hyhridoma cell lines, by immunization to elicit a polyclonal antibody response, or by recombinant host cells that have been transformed with a recombinant DNA expression vector that encodes the antibody or antibody fragment.
  • antigen is defined as a molecule that induces the formation of an antibody or is capable of binding specifically to the antigen-binding sites of an antibody.
  • biotinylation enzyme refers to the class of enzymes known as biotin protein ligases, or enzymes which biotinylate other proteins or peptides.
  • epipe refers to that portion of an antigen that interacts with an antibody.
  • host cell refers to a eukaryotic cell or group of cells that can be or has been transformed by a recombinant DNA vector.
  • operably linked refers to the placement of one nucleic acid into a functional relationship with another nucleic acid.
  • a promoter is “operably linked” to a coding sequence if the promoter causes the transcription of the coding sequence.
  • operably linked means that the DNA sequences being linked are contiguous and, where necessary to join two peptide or protein coding regions, in reading frame with one another.
  • peptide refers to an oligomer in which the monomers are amino acids (usually alpha-amino acids) joined together through amide bonds.
  • a "peptide” can be referred to as a "polypeptide.”
  • Peptides are more than two amino acid monomers long, but more often are more than 5 to 10 amino acid monomers long and can be even longer than 20 amino acids, although peptides longer than 20 amino acids are more likely to be called “polypeptides”.
  • the term “protein” is well known in the art and usually refers to a very large polypeptide, or set of associated polypeptides, that has some biological function. For purposes of the present invention the terms "peptide,” “polypeptide,” and “protein” are largely interchangeable as libraries of all three types can be prepared using substantially similar methodology.
  • recombinant DNA cloning vector and “recombinant DNA expression vector” refer to a DNA or RNA molecule that encodes a useful function and can either be used to transform a host cell or be introduced into a cell-free translation system to produce a protein encoded by the vector.
  • a cloning vector typically serves primarily as an intermediate in the construction of an expression vector; the latter vector is used to transform or transfect a host cell (or is introduced into a cell-free transcription and translation system) so that the transformed host cell (or cell-free transcription and translation system) produces a protein or other product encoded by the vector.
  • Such vectors are typically "plasmids," which, for purposes of the present invention, are vectors that can be extrachromosomally maintained in a host cell, but can also be vectors that integrate into the genome of a host cell.
  • FIG. 1 Illustration of 100 % biotinylation of GFP (green fluorescent protein) in vivo with different BAD vectors.
  • HEK293 cells transduced with pBBHN.TAP54 vector, were transiently transfected with a pBN.GFP plasmid (expressing GFP BAD-domain fusion without BirA). They were further analysed as in 1 A.
  • HEK 293 cells were transiently cotransfected with pBN.GFP and CMV.BirA. They were further analysed as in 1 A. - Figure 3 : Demonstration that heterochromatin protein 1 (HPl) localization is not disturbed by in vivo biotinylation.
  • HPl heterochromatin protein 1
  • HPl localization 3T3 cells were transfected with pBBHN.HPl ⁇ and double- stained with anti-HP l ⁇ antibodies and streptavidin, detected by Rhodamin or FITC, correspondingly. Chromatin was stained with Hoechst 33258. The pattern of staining of the cells with HPl is exactly the same between chromatin, biotinylated HPl and endogenous HPl.
  • EBP Endogenous Biotinylated Proteins (acetylCoA carboxylase, pyruvate carboxylase).
  • 3T3 cells were transfected with pBBHN.HPla and the extract was analysed on western with antiHPl antibodies and with streptavidin-HPO. The endogenous HPl and the biotinylated HPl are indicated.
  • the vector containing the pattern LTR- ⁇ -protein of interest-IRES-BirA-LTR leads to the production of viral particles containing the DNA of interest.
  • the inventors constructed retroviral vectors that allow to insert an open reading frame of a protein of interest to express a fusion version of this protein, that bear, as a N- or C-terminal tag, a sequence that is efficiently biotinylated by a BirA enzyme. They designed two different versions of the vector that can be used for either N- terminal or C-terminal tagging. In another version of the same vectors they added a His tag, to use for double-epitope tag purification.
  • the ORF for the BirA enzyme is placed on the same vector downstream of the gene of interest and its expression from the same mRNA is ensured by an Internal Ribosome Entry Sequence (IRES).
  • IRS Internal Ribosome Entry Sequence
  • This design allows to provide a high local concentration of the BirA enzyme in the proximity of the protein of interest, which would ensure efficient biotinylation with minimal amounts of the BirA enzyme.
  • An additional feature of this particular set of vectors is the third ORF encoding a selection marker IL2R, which is under control of a second IRES. This tricistronic design allows to minimize the number of steps and reagents necessary to generate the required cell line.
  • the GFP is in the form that can strongly interact with streptavidin : this is in the medium without exogenous biotin (streptavidin +, biotin -). However, when the cells are incubated in the biotin containing medium, 100 % of the GFP is in the form that strongly interacts with streptavidin. In summary, they achieved 100 % biotinylation in this system.
  • the vector pBBHN used referenced SEQ ID N°l in the sequence listing (Retroviral vector for creation of stable cell lines expressing biotinylated protein of interest), comprises 9037 nucleotides as follows :
  • 606-2872 mouse genomic DNA, LTR, splice donor, packaging, pol-gag, splice acceptor sequences of the pMFG retroviral vector (constructed by Paul Robbins and
  • 2956-2975 Xhol, Nsil, Notl restriction sites containing polylinker.
  • IRES 1 is a sequence described in the paper by Chappell et al Edelman GM, Mauro VP. (Proc Natl Acad Sci U S A. 2000 Feb 15;97(4):1536-41referenced SEQ ID N°2 in the sequence listing). It is a relatively short sequence that is, presumably, recognised by a 18S ribosomal RNA.
  • EMC IRES EMC IRES (IRES from encephalomyocarditis virus, widely used in the field for expression of polycistronic mRNA in mammalian cells. For example, see Morgan RA et al Nucleic Acids Res. 1992 Mar 25;20(6): 1293-9.)
  • 4636-5454 IL2R ORF (alpha subunit of the the InterLeukin 2 Receptor. Its use for the magnetic affinity sorting first described in Padmanabhan R et al Anal
  • FIG. 7 Identification of interaction partners of the protein of interest. 293 cell lines were generated expressing biotinylated versions of the proteins TAP54 ⁇ and HPl ⁇ which were also carrying a 6XHis tag on their N terminus. The complexes of these proteins with their interaction partners were purified from the total cell extract in two step affinity chromatography, using IMAC chromatography and immobilized streptavidin chromatography for the first and second step, correspondingly (see materials and methods). As seen in the Figure 7, proteins specific for the TAP54 ⁇ sample are represented by a doublet approximately 50 kd size. Proteins specific for the HPl ⁇ sample are represented by doublets of 28 kd and 30 kd size.
  • TAP54 ⁇ specific bands were excised and processed for mass- spectrometrical analysis. Both upper and lower bands yielded peptides with sequence GTEVQVDDIK, GLGLDDALEPR, which identified them as TAP54 ⁇ , the upper band apparently being the epitope-tagged TAP54 ⁇ and the lower band the endogenous TAP54 ⁇ .
  • the lower band also contained peptide with the sequence KTEVLMENFR, which corresponds to TAP54 ⁇ .
  • 293 cells were cotransfected with CMV.BBHN.TAP54 ⁇ and ⁇ OZ.FHTAP54 ⁇ , expressing biotinylation version of TAP54 ⁇ and HA-tagged version of its interaction partner TAP54 ⁇ , correspondingly.
  • Total cell extract from transfected cells was subjected to immunoprecipitation with immobilised streptavidin to pull down the
  • TAP54 ⁇ and the presence of TAP54 ⁇ was followed by western with anti-HA antibodies.
  • detection of HA- TAP54 ⁇ required the presence of biotinylated TAP54 ⁇ in the sample, consistent with interaction of these two proteins.
  • a mutation in TAP54 ⁇ (TAP54 m) abolishing its ATPase activity, was binding with TAP54 ⁇ without apparent difference from the wild type.
  • Figure 8A corresponds to analysis of the samples before immunoprecipitation.
  • Biotinylation system allows to use more stringent washing conditions to study the protein-DNA interactions in vivo.
  • H2AZ is a replacement histone shown to be enriched in active chromatin, but also present in other parts of genome (Redon C and al., Curr Opin Genet Dev. 2002 Apr; 12(2): 162-9. Review). Chromatin from NIH3T3 cells expressing biotinylated H2AZ was used for chromatin immunoprecipitation with immobilized streptavidin, and washing conditions of various stringency were tested (see materials and methods). To quantify the presence of mouse genomic DNA in the samples, quantitative PCR to amplify repetitive Bl sequences (randomly distributed throughout the genome) were used.
  • chromatin from the cells expressing biotinylated transcription factor DPI (DRTF1 -polypeptide- 1; DRTF1 for cell cycle transcription factor) was used and DHFR promoter was tested, a known DNA target of this protein.
  • DPI biotinylated transcription factor
  • the chromatin from the DPI expressing cells yielded significantly more DNA corresponding to the DHFR promoter compared to the chromatin from GFP expressing cell lines.
  • PCR with irrelevant fragment, covering the promoter of inactive myogenin gene did not show any difference between the control and experimental sample (bottom panel). This demonstrates that this procedure is detecting specific interactions between the protein of interest and DNA.
  • 2 % SDS washing step improved the signal to noise ratio (not shown).
  • Figures 7 to 10 demonstrate that the invention allows identification of the interaction protein partners of the protein of interest by purifying a complex of biotinylated protein with other proteins and identifying them by mass-spectromtery, that the method according to the present invention can be used to follow in vivo interaction between different proteins using coimmunoprecipitation, and that this method has an advantage in the chromatin immunoprecipitation experiments, as it affords more stringent washing conditions and significally lowers the nonspecific background.
  • DMEM Dulbecco's modified Eagle's medium
  • FCS 10 % fetal bovine serum
  • retroviral transduction first packaging cell line Phenix-E was transfected with the plasmid DNA of the retroviral vector, in two days the retroviral supernatant was harvested, filter sterilised and added to the HeLa or HEK 293 cells. Two days after, the transduced cells were sorted using magnetic affinity beads coupled with anti-IL2R antibodies (Ogryzko and all, Cell 1998, 94, 35-44).
  • the SDP-PAGE separation, transfer to a nitrocellulose membrane, blocking, incubation with antibody and ECL detection are according to a standard protocol, except that for the detection of biotinylated proteins the washes were performed in a more stringent conditions by adding 500 mM NaCl to the washing buffer (PBS + 0.1 % Tween).
  • the anti-GFP antibodies are from Clontech.
  • the HPl antibodies are from Euromedex.
  • the streptavidin-HPO conjugate is from Sigma. Immunofluorescence :
  • NIH3T3 cells were grown and transfected on coverslips. Fixation was with 4 % formaldehyde in PBS for 15 min at 4 C. Fixed cells were permeabilised with 0.5 % Triton for 15', blocked with 1 % BSA and 10 % FBS and incubated with anti HPlg antibodies. Then they were washed with PBS and incubated with secondary antibody conjugated to rhodamine. For detection of biotinylated proteins the cells were incubated with streptavidin-FITC for 1 hour at 37C and washed with PBS. The cells were observed on the fluorescent microscope and the images were taken using a CCD camera.
  • 293 cells were transfected using calcium phosphate method in one well of a 6 well plate. Two days after transfection, the cells were harvested, washed with PBS and extracted with Buffer B. The extract was incubated with streptavidin bound magnetic beads for 1 hour and washed 5 times with the buffer B. After, the beads were boiled in SDS sample buffer and processed for Western analysis. Chromatin immunoprecipitation and PCR analysis : Cells were transfected with polyfect, according to the supplied protocol (Qiagen, cat. 301105). Two days after transfection, 37 % formaldehyde (Sigma,) was added to the culture medium to a final concentration of 1 %.
  • Crosslinking was performed at 37 °C for 10 minutes and stopped by addition of glycin to a final concentration of 0.125 M, followed by a 5 minutes incubation at room temperature.
  • Fixed cells were washed twice with cold PBS and harvested in cold PBS containing protease inhibitors. Cells were pelleted by centrifugation, resuspended in 1 mL of SDS buffer (50 mM Tris at pH 8.1, 0.5 % SDS, 100 mM NaCl, 5 mM EDTA and protease inhibitors) and incubated 10 minutes on ice.
  • SDS buffer 50 mM Tris at pH 8.1, 0.5 % SDS, 100 mM NaCl, 5 mM EDTA and protease inhibitors
  • IP buffer 0.3 % SDS, 1.1 % Triton X100, 1.2 mM EDTA, 16.7 mM Tris at pH 8.1, 167 mM NaCl, and protease inhibitors
  • 50 mg of chromatin was diluted in a final volume of 300 mL of IP buffer.
  • Chromatin was precleared 1 hour by addition of 40 mL of blocked protein A beads (Pierce, cat.
  • Samples were then decrosslinked by an overnight incubation at 67 °C in 6O mL of 300 mM NaCl solution. Proteinase K and 5X Proteinase K buffer (50 mM Tris at pH 7.5, 25 mM EDTA, 1.25% SDS) were then added to the samples. After a 2 hour incubation at 45 °C, DNA from the samples was purified using Qiagen miniprep columns (cat. 27106). Purified DNA was recovered in 50 mL of water.
  • biotinylation does not generally disturb ability of a protein to interact with other proteins, and that biotinylated HPl proteins colocalize with heterochromatin and with endogenous HPl proteins, while other proteins, that are not expected to be in heterochromatin, show a different localization pattern.
  • biotinylated TAP54 extracted from cells shows that it is present in large complexes. Further, expression levels of the biotinylated proteins of interest are usually lower than the levels of the endogenous proteins.
  • the inventors have made Western analysis of HPl and MyoD expression.
  • the inventors have also shown, in comparison with the methods of the prior art, that purification of a protein complexes using biotinylation is more efficient, and that detection of nucleic acids (DNA) associated with biotinylated proteins is more efficient and more specific.
  • DNA nucleic acids
  • the invention provides the main following advantages compared to the state of the art.
  • the invention addresses an additional problem particular to the large eukaryotic cells - how to minimize potential negative impact of expression of the heterologous enzyme BirA in the mammalian cells, at the same time ensuring the 100 % biotinylation of the desired protein.
  • the inventors solve it by coexpressing the biotinylating enzyme from the same mRNA, thus increasing its local concentration exactly where it is needed (this allows to minimize the expression of the BirA enzyme, required to achieve 100 % biotinylation of the protein).
  • this polycistronic design has an additional advantage, as it also minimises the time and effort necessary for generation of the recombinant mammalian cells.
  • the advantages of the epitope tagging technique are practically the same as those of the native complex purification, as this system is the closest to probing the actual macromolecular interactions in the cell.
  • it is easier to adapt to the high throughput format, as it overcomes the limitations of the native complex purification approach, rooted in the antibody development and use.
  • the protocol is significally less expensive, fast and easily standardized, as it requires only one antibody and the cDNA, instead of a protein.
  • epitope tags Flag-, HA-, myc-tags etc
  • the corresponding antibodies are commercially available.
  • Nonspecific binding of undesired material (proteins/DNA) to the chromatographic medium during complex purification can be controlled by comparing the results of the purification with so called mock purification, i.e. incubation of the same medium (antibodies, beads etc) with an extract from the parent cells that do not express the epitope-tagged protein. Similar control is difficult in the case of antibodies against native proteins, as it would require finding cells that do not express the protein.
  • biotinylation technique used by the inventors allows : a. Faster biochemical purification
  • the inventors use one vector to generate in one step mammalian cell lines expressing biotinylated proteins.
  • the one vector approach saves time and effort.
  • retroviral vector to generate cell lines expressing biotinylated proteins.
  • the retroviral vectors also minimizes time and the efficiency of gene transfer is much higher compared to the most other vectors.
  • the IRES used minimize the amounts of the BirA enzyme necessary for 100% biotinylation of the target protein. This deals with the problem of negative effect of the BirA overexpression on the cell physiology and of biotinylation of undesirable proteins that increases the background during complex purification.
  • the particular IRES used in the vector is smaller than other IRESes, commonly used (40 bp instead of 500). It helps to maximize the capacity of the retroviral construct and in general, minimize the vector size.
  • Most of potential applications relate to fast and efficient creation of mammalian cell lines that express a biotinylated version of protein of interest.
  • These cell lines then can be used for detection, localisation, and purification of the proteins of interest directly from mammalian cells. Also, these cell lines can be used for identification of interaction partners of the proteins of interest by affinity chromatography on streptavidin column. It can be done directly either from cellular (nuclear) extracts or lysates, from native or from crosslinked (in vivo or in vitro) material.
  • the invention shows significantly improved results in namely the detection of proteins, their localization in vivo (by fluorescent or electron microscopy), their labeling, the identification of interaction partners (proteins and nucleic acids) in vivo (by coimmunoprecipitation on immobilized streptavidin or avidin), the visualization of set of interaction partners.
  • the invention allows an expression of efficiently biotinylated proteins, an expression of efficiently postranslationally modified proteins that require the modification for their function (phosphorylated, acetylated, methylated, ubiquitinated, sumoilated, protease cleaved, etc).

Abstract

Procédé pour exprimer dans des cellules eucaryotes une protéine cible d'intérêt modifiée qui consiste à exprimer (i) une protéine d'intérêt et (ii) une enzyme de modification qui permet la modification de ladite protéine cible, la protéine cible d'intérêt et l'enzyme de modification étant coéxprimées à partir du même ARN.
EP03756089A 2002-05-30 2003-05-30 Vecteurs destines a l'expression de proteines biotinylees dans des cellules mammaliennes et leur utilisation pour identifier les interactions proteines - acides nucleiques in vivo Withdrawn EP1513935A1 (fr)

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EP20020291316 EP1367125A1 (fr) 2002-05-30 2002-05-30 Vecteurs pour l'expression de protéines biotinylées dans des cellules de mammifères et leur utilisation pour identifier des interactions entre protéines et acides nucléiques in vivo
EP03756089A EP1513935A1 (fr) 2002-05-30 2003-05-30 Vecteurs destines a l'expression de proteines biotinylees dans des cellules mammaliennes et leur utilisation pour identifier les interactions proteines - acides nucleiques in vivo
PCT/IB2003/002899 WO2003102196A1 (fr) 2002-05-30 2003-05-30 Vecteurs destines a l'expression de proteines biotinylees dans des cellules mammaliennes et leur utilisation pour identifier les interactions proteines - acides nucleiques in vivo

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EP03756089A Withdrawn EP1513935A1 (fr) 2002-05-30 2003-05-30 Vecteurs destines a l'expression de proteines biotinylees dans des cellules mammaliennes et leur utilisation pour identifier les interactions proteines - acides nucleiques in vivo

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