WO2011003896A1 - Detection and visualization of the cell cycle in living cells - Google Patents
Detection and visualization of the cell cycle in living cells Download PDFInfo
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- WO2011003896A1 WO2011003896A1 PCT/EP2010/059622 EP2010059622W WO2011003896A1 WO 2011003896 A1 WO2011003896 A1 WO 2011003896A1 EP 2010059622 W EP2010059622 W EP 2010059622W WO 2011003896 A1 WO2011003896 A1 WO 2011003896A1
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- nucleic acid
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6875—Nucleoproteins
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
Definitions
- the present invention relates to a nucleic acid molecule encoding a polypeptide specifically binding to proliferating cell nuclear antigen (PCNA), said nucleic acid molecule comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO 2 or an amino acid sequence deviating from SEQ ID NO 2 by conservative substitution of one or more amino acids in position 1 to 28, 38 to 52, 63 to 98 and 115 to 123 of SEQ ID NO 2
- the present invention also relates to a vector comprising the nucleic acid molecule of the invention, a host cell comprising the nucleic acid molecu ⁇ e of or the vector of the invention and a method of detecting the amount and/or location of PCNA in living cells, as well as a method of screening for compounds having an effect on the cell cycle
- Antibodies are valuable tools to identify and visualize cellular structures
- the application of naturally occurring antibodies for the detection of intracellular antigens requires permeabilization (and often fixation) of cells
- the antibody-based detection of antigens within intact celis is essentially prevented by the fact that antibodies are, by nature, designed to function in an oxidizing (extracellular) environment the reducing environment sn the cytoplasm leads to an impaired disulfide bond formation, resulting in an inefficient assembly of epitope recognizing parts of the variable Sight and heavy chain (B'occa et a!
- the present invention relates to a nucleic acsd molecule encoding a polypeptide specifically binding to proliferating cell nuclear antigen (PCNA), said nucleic acid molecule comprising a nucleic acid sequence encoding the ammo acsd sequence of SEQ ID NO 2 or an amino acid sequence deviating from SEQ ID NO 2 by conservative substitution of one or more amino acids in position 1 to 28, 38 to 52, 63 to 98 and 115 to 123 of SEQ ID NO 2
- Examples of such polypeptides comprising conservative substitutions of one or more ammo acids in one or two framework regions are represented by SEQ ID NOs 16 and 18
- nucleic acid molecule as used interchangeably with the term “polynucleotide”, in accordance with the present invention, includes DNA such as cDNA or genomic DNA, and RNA such as mRNA Further included are nucieic acid mimicking molecules known in the art such as synthetic or semi-synthetic derivatives of DNA or RNA and mixed polymers as long as they can be introduced and expressed sn a cell Nucleic acid molecules may contain additional non-natural or derivative nucleotide bases as will be readily appreciated by those skilled in the art
- specifically binding' in accordance with the present invention refers to the property of the polypeptides of the invention to have an affinity for specific proteins, i e target protesns, which is greater by the factor of at least 10 preferably at least 100, more preferably ai ieasi 1000 and most preferably at least 10000 as compared to the affinity to proteins unrelated to the target protein
- the target protein of the polypeptide of the present invention is PCNA
- conservative substitution relates to replacement of one or more amino acids with another amino acid with similar chemical and physical properties such as size and charge
- conservative substitutions are the replacement of valine with leucine, of cysteine with serine, of giutamine with aparagine or of aspartic acid with glutamic acid or vice versa
- conservative substitutions do not or essentially not alter the properties of the polypeptide of the invention such as its binding properties and/or its behaviour within eukaryotic cells
- the term “essentially” denotes a binding activity of at least 70%, more preferably at least 80%, more preferably at least 90% and most preferably at least 95% as compared to that of the polypeptide of the invention not comprising amino acid substitutions
- the term "essentially” denotes e g an expression and/or distribution
- the amino acids from position 1 to 28, 38 to 52, 63 to 98 and 115 to 123 of SEQ ID NO 2 form the framework region of the polypeptide of the invention
- the framework regions as well as the CDR regions which are involved in the binding of PCNA have been determined (see Figure 1 )
- ammo acid substitutions are to be chosen such that they do not interfere with the correct folding of the polypeptide and do not alter the three dimensional structure of the framework so that binding to PCNA ⁇ S no longer possible
- ammo acids can be substituted and is also aware of methods for comparing the properties of a polypeptide comprising one or more ammo acid substitutions with those of the polypeptide of the invention not comprising any ammo acid substitutions
- Exempiary methods comprise solubility studies, aggregate formation assays distribution analyses, binding assays and expression studies in living eukaryotic cells
- nucleic acid of the invention encoding a polypeptide with one or more conservative substitutions on one or two framework regions are those of SEQ ID NOs 15 and 17 encoding the polypeptides of SEQ ID NOs 16 and 18 (see figure 9)
- the present inventors have ⁇ urp ⁇ singiy found that a PCNA-binding polypeptide can be applied in living cells for the detection of the presence and/or location of PCNA in said cells
- the polypeptide of the invention is derived from cameiid antibodies, the general recombinant production and selection procedure of which is well known in the art (e g by phage display and recombinant techniques, see e g Hoogenboom et a! (1998), Pluckthun (1994), Verma et a!
- the nucleic acid molecule comprises or consists of (a) the nucleic acid sequence of SEQ ID NO 1 , 15 or 17, or (b) a nucleic acid sequence degenerate with respect to the nucleic acid sequence of (a)
- degenerate in accordance with the present invention refers to the degeneracy of the genetic code
- a triplet code of bases designates 20 amino acids and a stop codon Because four bases exist which are utilized to encode genetic information, triplet codons are required to produce at least 21 different codes
- the possible 4 3 possibilities for bases in triplets gives 64 possible codons, meaning that some degeneracy must exist
- some amino acids are encoded by more than one triplet, i e by up to six
- the degeneracy mostly arises from alterations in the third position in a t ⁇ plet This means that nucleic acid molecuies having a different sequence than that specified above, but still encoding the same polypeptide he withsn the scope of the present invention
- visually detectable relates to a physical or chemical property of a marker that enables for its detection using visual means Typica ⁇ y, visually detectable markers can be detected since they emit radiation such as fluorescence, luminescence, phosphorescence or radioactivity
- the nucleic acid of the invention encodes a polypeptide comprising a polypeptide specifically binding to PCNA according to the invention (a first polypeptide component), a visually detectable peptidic or protesnaceous marker ⁇ a second polypeptide or peptidic component) such as a fluorescent polypeptide and a linker
- a polypeptide of this structure is, in connection with the present invention also termed "fusion protein"
- a fusion protein according to the invention comprises two polypeptide components or one polypeptide and one peptidic or proteinaceous component and a linker
- said visually detectable peptidic or proteinaceous marker is a fluorescent polypeptide, wherein said fluorescent polypeptide is separated from said polypeptide specifically binding to PCNA by a linker of at least one amino acid residue
- polypeptide as used herein interchangeably with the term “protein” describes linear molecular chains of amino acids, containing more than 30 amino acids Polypeptides according to the invention, in particular fusion proteins comprising a fluorescent polypeptide, preferably do not form oligomers
- polypeptide and “protein” also refer to naturally modified polypeptides/protems where the modification ;s effected e g by glycosylation acetylation, phosphorylation and similar modifications which are well known in the art
- fluorescent polypeptide or "fluorescent protein” refers to polypeptides emitting fluorescent lsght upon excitation at a specific wavelength
- GFP Green Fluorescent Protein isolated from Aequorea victoria
- these variants or the nucleic acids encoding them share about 80%, or greater sequence identity with the amsno acid sequence of SEQ iD No 4 or the nucleic acid sequence of SEQ ID NO 3, respectively
- Color-shift GFP mutants have emission colors blue to yellow-green, increased brightness, and photostability (Tsien, 1998)
- eYFP Enhanced Yellow Fluorescent Protein
- Another group of fluorescent proteins includes the fluorescent proteins isolated from anthozoans, sncludi ⁇ g without limitation the red fluorescent protein isolated from Dtscosoma species of coral, DsRed (Matz et a! , 1999) (see, e g , accession number AF168419) DsRed and the other anthozoan fluorescent proteins share only about 26-30% amino acid sequence identity to the wild-type GFP from Aequorea victoria, yet all the crucial motifs are conserved, indicating the formation of the 11- stranded beta-barrel structure characteristic of GFP The crystal structure of DsRed has also been solved, and shows conservation of the 11 -stranded beta-barrel structure of GFP (MMDB Id 5742) A number of mutants ot the longer wavelength red fluorescent protein DsRed have also been desc ⁇ bed, and similarly, may be employed in the generation of the fusion proteins of the invention comprising fluorescent (poly)pept ⁇ des For example, recently described DsRed mutant
- Monome ⁇ c versions of DsRed are e g mRFP, mCherry (e g having the amino acid sequence of SEQ ID NO 6 or encoded by the nucleic acid sequence of SEQ ID NO 5) mRFP1 (Campbell et al , 2002) mOrange or mPlum (Shaner et al , 2004) or TagRFP (Merzlyak et ai , 2007)
- GFPs from the anthozoans Renilla reniformis and Renilla kolliken were described (Ward et al , U S Patent Appn 20030013849), which may also be used in accordance with the invention
- An increasingly large number of other fluorescent proteins from a number of ocean life forms have recently been described, and the Protein Data Bank currently lists a number of GFP and GFP mutant crystal structures, as well as the crystal structures of various GFP analogs
- Related fluorescent proteins with structures inferred to be similar to GFP from corals, sea pens, sea squirts, and sea anemones have been described, and may be used in the generation of the fusion proteins of the invention (for reviews, see Zhang et al , 2002 Zimmer 2002),
- Fluorescent proteins from Anemonia majano, Zoanthus sp , Discosoma striata, Discosoma sp and Ciavularia sp have also been reported (Matz et al , 1999)
- a fluorescent protein cloned from the stony coral species, Trachyphyllia geoffroyi has been reported to emit green, yellow, and red light, and to convert from green light to red light emission upon exposure to UV light (Ando et a!
- fluorescent proteins from sea anemones include green and orange fluorescent proteins cloned from Anemonia sulcata (Wiedenmann et al 2000), a naturally enhanced green fluorescent protein cloned from the tentacles of Heteractis magnifica (Tu et al , 2003), a non- ohgome ⁇ zing red fluorescent protein based on the chromoprotein hcriCP from Heteractis c ⁇ spa (Gurskaya et al , 2001 , Fradkov et al , 2002) a generally non fluorescent purple chromoprotein displaying weak red fluorescence cloned from Anemonia sulcata and a mutant thereof displaying far- red shift emission spectra (595nm) (Lukyanov et al , 2000)
- Any fluorescent or chromophoric protesn or fluorescent or chromophoric fragment thereof may be used in accordance with the teaching of the present invention
- linker refers to the connection between the polypeptide components, or the polypeptide and the peptidic or proteinaceous components of the fusion protein of the invention
- a linker in accordance with the present invention is comprised of amino acids comprising at least one ammo acid residue which is located between the mentioned polypeptide components, or the polypeptide and the peptidic or proieinaceous components of the fusion protesn
- Such a linker may in some cases be useful, for example, to improve separate folding of the individual polypeptide components of the fusion protem of the invention or to modulate the stability of the fusion protein
- linker residues may contain signals for transport, protease recognition sequences or signals for secondary modification
- the linker may be as short as 1 ammo acid residue or up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 40 or 50 residues in particular cases, the linker may even involve up to 100 or 150 residues It is preferred that the linker comprises between 18 and 30 amino acids, more preferably between 20 and 25 amino acids and most preferably between
- Particularly preferred linkers comprise or consist of the amino acid sequence of SEQ ID NOs 13 or 14
- the fluorescent polypeptide is GFP, RFP or YFP, preferably encoded by the nucleic acid sequence of SEQ ID NO 3, 5 or 7 or having the amino acsd sequence of SEQ ID NO 4, 6 or 8, or a fluorescent mutant or fragment thereof
- a "fluorescent mutant” in accordance with the present invention denotes any fluorescent protein, preferably derived from the above GFP, RFP or YFP sequences Fluorescent mutants preferably have a sequence identity of at least 60%, more preferably at least 70%, even more preferably at least 80% most preferably at least 85%, such as at least 90%, at least 95% or 100% to said GFP, RFP or YFP while retaining or essentially retaining their fluorescent properties
- a "fluorescent fragment” according to the invention relates to parts or fragments of the above GFP, RFP or YFP or fluorescent mutants thereof which are shortened in their sequence but still retain or essentially retain their fluorescent properties
- the term “essentially retain” refers to the fluorescent properties of fluorescent mutants or fluorescent fragments according to the invention
- fluorescent properties essential in context with the present invention while in principle also referring to absorption or emission spectra, relate to the brightness of a fluorescent mutant or fluorescent fragment as compared to the fluorescent polypeptide from which it is derived
- the term “essentially retain” means that the fluorescent mutant or fluorescent fragment has at least 50%, preferably at least 60%, more preferably at ieast 70%, even more preferably at least 80%, yet more preferably at least 90% most preferably at least 95% of the brightness of the fluorescent polypeptide from which it is derived in another more preferred embodiment the polypeptide specifically binding to PCNA is located N- terminally of said fluorescent polypeptide
- the polypeptide specifically binding to PCNA may sn principle also be located C-terminally of said fluorescent polypeptide, the appended examples have been carried out with the polypeptide specifically binding to PCNA being located N-termmally of the fluorescent polypeptide The functionality of this arrangement is
- nucieic acid molecule comprises or consists of the nucleic acsd sequence of SEQ ID NO 9, 11 , 19 or 21 or encodes an amino acid sequence comprising or consisting of SEQ !D NO 10, 12 20 or 22
- the polypeptide of the invention specifically binding to PCNA when fused to GFP or RFP, not only exerts the desired binding activity to PCNA but can also be expressed intracellulariy without interfering with the cellular processes involving PCNA This enables for a reliable detection of the presence and/or location of PCNA in the living cell
- the present invention relates to a (expression) vector comprising the nucleic acid molecule of the invention
- the vector is a plasmid, cosmid, virus, bacteriophage or another vector used conventionally e g in genetic engineering
- the nucleic acid molecule of the present invention may be inserted into several commercially available vectors
- Non-limiting examples include prokaryottc plasmid vectors, such as the pUC- se ⁇ es, pBluescript (Stratagene ⁇ , the pET-se ⁇ es of expression vectors (Novagen) or pCRTOPO (Invitrogen), lambda gt11 , pJOE, the pBBR1-MCS series, pJB861 , pBSMuL, pBC2, pUCPKS, pTACTI and vectors compatible with expression in eukaryotic cells, such as yeast or mammalian cells, like pREP (Invitrogen), pCEP4 (tnvitrogen), pMCI neo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2neo, pBPV-1 , pd
- the nucleic acid molecule of the present invention referred to above may also be inserted into vectors such that a (further) translational fusion with another nucleic acid molecule is generated
- the other nucleic acsd molecules may encode a protein which may e g increase the solubility and/or facilitate the pu ⁇ fcation of the protein encoded by the nucieic acid molecule of the invention
- Non- limitmg examples include pET32, pET41 , pET43
- the vectors may also contain an additional expressible polynucleotide coding for one or more chaperones to facilitate correct protein folding
- Suitable bacterial expression hosts comprise e g strains derived from BL21 (such as BL21(DE3), BL21 (DE3)PlysS, BL21(DE3 ⁇ R!L, BL21(DE3)PRARE) or Rosetta ⁇
- vectors can contain one or more origins of replication (on) and inheritance systems for cloning or expression, one or more markers for selection in the host, e g , antibiotic resistance, and one or more expression cassettes
- origins of replication include, for example, the CoI E1 , the SV40 viral and the M 13 origins of replication
- the coding sequences according to the invention inserted in the vector can e g be synthesized by standard methods, or isolated from natural sources Ligation of the coding sequences to transcriptional regulatory elements and/or to other amino acid encoding sequences can be carried out using established methods
- Transcriptional regulatory elements parts of an expression cassette
- These elements comprise regulatory sequences ensuring the initiation of transcription (e g , translation initiation codon, promoters, enhancers, and/or insulators ⁇ , internal ribosomal entry sites (IRES) (Owens ef a/ , 2001 ) and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript
- Additional regulatory elements may include transcriptional as well as translational enhancers, and/or naturally-associated or heterologous promoter regions
- the nucleic acid molecule of the invention is operably linked to such expression control sequences allowmg expression sn prokaryo
- An expression vector according to this invention is capable of directing the replication, and the expression of the nucleic acid molecule of the invention and the polypeptide encoded thereby
- the nucleic acid molecules of the invention as described herein above may be designed for direct introduction or for introduction via liposomes, phage vectors or viral vectors (e g adenoviral, retroviral) into the cell Additionally, baculoviral systems or systems based on Vaccinia Virus or Semliki Forest Virus can be used as vector in eukaryotic expression system for the nucleic acid molecules of the invention
- Expression vectors derived from viruses such as retroviruses, vaccinia virus adeno-assoctated virus, herpes viruses or bovine papilloma virus may be used for delivery of the polynucleotides or vector into targeted cell population Methods which are well known to those skilled in the art can be used to construct recombinant viral vectors see, for example, the techniques described in Sambrook, 2001 and Aus
- a typical mammalian expression vector contains the promoter element, which mediates the initiation of transcription of mRNA, the protein coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript Moreover, elements such as origin of replication, drug resistance gene, regulators (as part of an inducible promoter) may also be included Additional elements might include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA sphcing Highly efficient transcription can be achieved with the early and late promoters from SV40, the long terminal repeats (LTRs) from retroviruses, e g , RSV, HTLVI, HIVI, and the early promoter of the cytomegalovirus (CMV) However, cellular elements can also be used ⁇ e g , the human acti ⁇ promoter) Other examples for regulatory elements permitting expression in eukaryotic host cells (the more preferred embodiment) are the A0X1 or G AU promoter in yeast or the chicken beta-actin promoter
- the co-transfection with a selectable marker such as dhfr, gpt neomycin, hygromy ⁇ n aliows the identification and isolation of the transfected cells
- the transfected nucleic acid can also be amplified to express large amounts of the encoded (poly)pept ⁇ de
- the dhfr (dihydrofoiate reductase) marker is usefu!
- GS giutamine synthase
- Suitable expression vectors for Drosophila are those belonging to the pMT DES system (invitrogen) using the drosophsla metallothionein (MT) promoter (Bunch et ai , 1988) or pAC5 1 using the drosophila actin 5C promoter
- MT drosophsla metallothionein
- pAC5 1 using the drosophila actin 5C promoter
- GAL4- ⁇ nduc ⁇ ble USA promoter is pUAST Yeast vectors are the pYEp vector (using a GaH O promoter), pYX142 (single copy vector) or pYX232 (2 ⁇ plasmid using the TP! t ⁇ osephosphat isomerase promoter (both Novagen ⁇ )
- Possible regulatory elements permitting expression in prokaryotic host cells comprise, e g , the lac, trp or tac promoter, the lacUVS or the trp promoter in E coli
- Suitable seiectabie markers in prokaryotic cells are tetracycline, kanamycm or ampicillin resistance genes for culturing in E coli and other bacteria in another aspect, the present invention relates to a host ceil comprising the nucleic acid molecule or the vector of the invention
- Hosts according to the invention can be single cells or non-human multi-cellular organisms
- Suitable proKaryotic hosts comprise e g bacteria of the species Escherichia, Streptomyces, Salmonella or Bacillus
- Suitable eukaryotic host cells are e g yeasts such as Saccharomyces cerevisiae or Pichia pastoris
- Insect cells suitable for expression are e g Drosophila S2 or Spodoptera Sf9 cells
- Mammalian host cells that couid be used include, human HeIa, HEK293, H9 and Jurkat cells mouse NIH3T3 and C127 cells, COS 1 , COS 7 and CV1 , quail QC 1-3 cells, mouse L cells, Bowes melanoma cells and Chinese hamster ovary (CHO) cells
- primary mammaltan cells or cell lines Primary ceils are cells which are directly obtained from an organism Suitable primary cells are, for example, mouse embryonic fibroblasts (MEF), mouse primary hepatocytes, cardiomyocytes and neuronal cells as well as mouse muscle stem cells (satellite celis) and stable, immortalized cell lines derived thereof
- the nucleic acid molecule of the invention can be expressed sn stable cell hnes that contain the nucleic acid molecule of the invention, preferably the vector of the invention integrated into a chromosome
- Transgenic non-human animals as hosts transfected with an ⁇ /or expressing the nucleic acid molecule of the present invention also lie within the scope of the invention
- the transgenic animal s a mamma!, e g a hamster, mouse rat, cow, cat pig, dog, horse, rabbit or monkey
- a method for the production of a transgenic non-human animal comprises introduction of the nucleic acid molecule or targeting vector of the present invention into a germ cell, an embryonic cell, stem ceil or an egg or a cell derived therefrom
- the non-human animal can be used in accordance with the invention e g in a method for identification of compounds, described herein below
- Production of transgenic embryos and screening of those can be performed, e g , as described by Joyner (1993)
- the DNA of the embryonic membranes of embryos can be analyzed using, e g , Southern blots with an appropriate probe, see supra
- a general method for making transgenic non-human animals is described in the art, see for example WO 94/24274
- embryonal stem cells are preferred Murine ES cells, such as AB-1 line grown on mitotica ⁇ ly inactive SNL76/7 cell feeder layers (McMah
- the success of generating a mouse line from ES cells bearing a specific targeted mutation depends on the pluripotence of the ES cells ( ⁇ e their ability, once injected into a host developing embryo, such as a blastocyst or morula to participate in embryogenesis and contribute to the germ cells of the resulting animal)
- the blastocysts containing the injected ES cells are allowed to develop in the uteri of pseudopregnant non-human females and are born, e g as chimeric mice
- the resultant transgenic mice are chimeric for celis having either the recombinase or reporter loci and are backcrossed and screened for the presence of the correctly targeted transgene (s) by PCR or Southern blot analysis on tail biopsy DNA of offspring so as to identify transgenic mice heterozyg
- Transgenic plants as hosts transfected With and/or expressing the nucleic acid molecule of the present invention also lie within the scope of the present invention
- the present invention also relates to a method of producing a polypeptide comprising cultu ⁇ ng the host cell of the invention under suitable conditions and isolating the polypeptide produced
- Said host cell expresses or overexpresses the polypeptide of the invention specifically binding to PCNA as described above Accordingly said polypeptide of the invention is produced in and isolated from the host cell
- Suitable conditions for culturing a prokaryotic or eukaryotfc host are well known to the person skilled in the art
- suitable conditions for culturing bacteria are growing them under aeration in Luna Bertani (LB) medium
- the medium can be buffered or supplemented with suitable additives known to enhance or facilitate both E coli can be cultured from 4 to about 37 0 C, the exact temperature or sequence of temperatures depends on the molecule to be overexpressed
- these conditions may have to be adapted to the needs of the host and the requirements of the polypeptide expressed in case an inducible promoter controls the nucleic acid of the invention in the vector present in the host cell, expression of the polypeptide can be induced by addition of an app ⁇ op ⁇ ate inducing agent
- Suitable expression protocols and strategies are known to the skilled person
- mammalian ceil culture which is a preferred embodiment for producing the polypeptide can e g be carried out tn RPMI or DMEM medium containing 10% (v/v) FCS, 2mM L-giutamine and 100 U/ml penicillin/streptomycine
- the ceils can be kept at 37 0 C in a 5% CO 2 , water saturated atmosphere
- Suitable expression protocois for eukaryotic cells are well known to the skilled person and can be retrieved e g from Sambrook, 2001
- Suitable media for insect cell culture is e g TNM + 10% FCS or SF900 medium Insect cells are usually grown at 27 °C as adhesion or suspension culture
- Methods of isolation of the polypeptide produced are well-known sn the art and comprise without limitation method steps such as ion exchange chromatography, gel filtration chromatography (size exclusion chromatography), affinity chromatography, high pressure liquid chromatography (HPLC), reversed phase HPLC 1 disc gel electrophoresis or immunoprecipitation, see, for example, in Sambrook, 2001
- the present invention relates to a polypeptide encoded by the nucleic acid molecule of the invention or produced by the method of producing a polypeptide according the invention
- the nucleic acid molecule comprises or consists of the nucleic acid sequence of SEQ ID NO 1 , 15 or 17 or a nucleic acid sequence degenerate with respect to SEQ ID NO 1 , 15 or 17
- the polypeptide of the invention further comprises a radioactive label Said radioactive label may be attached to the polypeptide during or after (recombinant) expression Radioactive labels comprised in the polypeptide of the invention enable for the rapid and immediate detection of the amount and/or location of said polypeptide in living ceils
- the present invention also relates to a polypeptide encoded by the nucleic acid molecule of the invention, preferably comprising or consisting of the nucleic acid sequence of SEQ ID NO 1 , 15 or 17 or a nucleic acid sequence degenerate with respect to SEQ ID NO 1 , 15 or 17, having a fluorescent dye coupled thereto
- Fluorescent dyes in accordance with the present invention are non-proteinaceous compounds which emit fluorescent light upon excitation
- Exemplary fluorescent dyes are those of the Alexa family ⁇ such as Alexa Fluor 350, 405, 430 488, 500, 514, 532 546, 555, 568, 594 610, 633, 647, 660, 680 700 and 750) the excitation and emission spectra of which cover the visible spectrum and extend into the infrared, HiLyte Fluors (AnaSpec), DyUght fluors (Pierce), the ATTO Dye series (ATTO-TEC and Sigma-Ald ⁇ ch) and also less bright dyes such as rhodamine and fluorescein
- Fluorescent dyes can be, for example, covalently coupled to primary amino groups of amino acsds, such as lysine, of proteins via N-Hydroxisuccinimid (NHS) ester formation
- polypeptide of the invention labeled with a fluorescent dye can be used e g for the detection of PCNA in immunofluorescence and immunoblotting applications Moreover it can be introduced into living cells using various techniques such as protein transduction (Schwarze et al , 2000) or protein delivery using for example cation ic lipids (proteofection) (Dalkara et al 2004) as will also be described further below in connection with nucleic acids
- the present invention relates to a method of producing a cell containing the nucle'C acid of the invention, comprising introducing the nucleic acid or the vector of the invention
- introducing a nucleic acid refers to the application of a nucleic acid to cells and its subsequent uptake and incorporation into the nucleus of said cells, preferably in the genetsc information or in the form of ⁇ ptsomes This definition can also be applied to vectors which are specific nucleic acids
- transfection For animal cells the process of introducing a nucleic acid 1 S cornrronly termed transfection, transformation or transduction If viruses are used, the process is termed infection or transduction Transfection of animal cells typically involves opening transient pores or 'holes' in the cell plasma membrane, to allow the uptake of material In addition to electroporation, transfection can be carried out by various methods of introducing foreign DNA into a ceil
- nucleofection heat shock, magnetofection and transfection reagents such as LipofectamineTM Dojsndo HilyMax Fugene, jetPESTM, Effectene or DreamFectTM it is preferred that the introduction of the nucleic acid is stable i e that it stably resides in the nucleus If the nucleic acid introduced does not itself encode a selectable marker which provides the cell with a selection advantage such as a resistance towards a certain herbicide toxin or antibiotic which is present in the growth medium of the cells and if the nucleic acid introduced is not stably incorporated into the nucleus, incorporation can be promoted by co-transformation with another nucleic acid encoding such a selectable marker
- the present invention relates to a method of detecting the amount and/or location of PCNA in iiving cells comprising (a) expressing in a eukaryotic eel! the polypeptide encoded by the nucleic acid molecule of the invention further encoding a fluorescent polypeptide and a linker of at least one ammo acid, and (b) detecting the fluorescence emission of the fluorescent part of said polypeptide in the cell upon excitation, wherein the location of the fluorescence emission of the fluorescent part of said polypeptide in the cell is indicative for the presence of PCNA
- the term "expressing in a eukaryotic cell” relates to the transcription and translation of the polypeptide encoded by the nucleic acid molecule of the invention further encoding a fluorescent polypeptide and a linker of at least one amino acid, i e the fus.on protem of the invention, using appropriate expression control elements that function in the chosen ceil in this manner the binding properties of individual fusion proteins according to the invention may be tested in cellular expression systems
- a nucleic acid molecule encoding a fusion protein may be cloned into a suitable expression vector as has been described above
- the expression system is eukaryotic preferably mammahan
- the present method simplifies basic research on the cell cycle by providing effective and easy-to-use means to detect the presence and in particular the location of the prominent eel! cycle marker PCNA
- the influence of e g different environmental conditions such as temperature, additives in the medium, radiation and any chemical compound on the cell cycle as well as the proliferation behaviour of different cell types
- the in vivo applicability of the polypeptide of the invention is useful in the screening for drugs having an influence on the cell cycle In particular it allows to study inhibitory as well as stimulating effects of drugs by determining the ongoing of the cell cycle in real tsme analysing the characteristic distribution of PCNA in the different cell cycle stages
- the polypeptide of the invention also distinct cell cycle stages (G1 -phase, S-phase, G2-pha ⁇ e or mitosis) can be distinguished by analysing the cellular distribution of the polypeptide which reflects the distribution of PCNA in the particular cell cycle stage
- the present invention also relates to a method of screening for compounds having an effect on the ceil cycle comprising (a) expressing in a eukaryotic cell the polypeptide encoded by the nucleic acid molecule of the invention further comprising a nucleic acid sequence encoding a fluorescent polypeptide, wherein said fluorescent polypeptide is separated from said polypeptide specifically binding to PCNA by a linker of at least one amino acid residue, (b) contacting the cell with a test compound, and (c) detecting the fluorescence emission of the fluorescent part of said polypeptide upon excitation, wherein a change in the amount and/or location of the fluorescence emission of the fluorescent part of said polypeptide in the cell as compared to that observed in a reference eukaryotic cell expressing the polypeptide encoded by the nucleic acid molecule of the invention further comprising a nucleic acid sequence encoding a fluorescent polypeptide and not contacted with the test compound is indicative that the compound has an effect on the cell cycle
- test compound can be but is not restricted to a compound belonging to the classes of nucleic acids, polypeptides peptides (amino acid sequences of up to 30 ammo acsds), peptide aptamers, nucleic acid based aptamers, ⁇ bozymes, small molecules or antibodies or fragments thereof
- the test compound can be any chemical compound
- a ⁇ bozyme (from ribonucleic acid enzyme, also called RNA enzyme or catalytic RNA) is an RNA molecule that catalyzes a chemical reaction Many natural ⁇ bozymes catalyze either their own cleavage or the cleavage of other RNAs, but they have also been found to catalyze the aminotransferase activity of the nbosome
- RNAs examples include the hammerhead, hairpin, hepatitis delta virus, and in vitro-seiected lead-dependent ⁇ bozymes
- the organization of these small catalysts is contrasted to that of larger ⁇ bozymes, such as the group I sntron
- Aptamers are oligonucietc acid or peptide molecules that bind a specific target molecule Aptamers are usually created by selecting them from a targe random sequence pool but natural aptamers also exist in ⁇ boswitches Aptamers can be used for both basic research and clinical purposes as macromoiecular drugs Aptamers can be combined with ⁇ bozymes to self-cleave in the presence of their target molecule These compound molecules have additional research, industrial and clinical applications More specifically, aptamers can be classified as DNA or RNA aptamers or peptide aptamers Whereas the former consist of (usua ⁇ y short ⁇ strands of oligonucleotides, the latter consist of a short va ⁇ able peptide domain, attached at both ends to a protein scaffold
- Nucleic acid aptamers are nucleic acid species that have been engineered through repeated rounds of in vitro selection or equivalently, SELEX (systematic evolution of Sigands by exponential enrichment) to bind to various molecular targets such as smail molecules, proteins, nucleic acids, and even cells, tissues and organisms
- Peptide aptamers are (poly)pept ⁇ des that are designed to interfere with other protein interactions inside cells They consist of a variable peptide ioop attached at both ends to a protem scaffold This double structural constraint greatly increases the binding affinity of the peptide aptamer to levels comparable to that of an antibody (nanomolar range)
- the va ⁇ able loop length is typically comprised of 10 to 20 ammo acids, and the scaffold may be any protein, which has good solubility properties
- the bacterial protein Th ⁇ oredoxin-A ss the most frequently used scaffold protem, the variable loop being inserted within the reducing active site, which is a -Cys-Gly-Pro-Cys- loop in the wild protein, the two cystein lateral chains being able to form a disulfide bridge Peptide aptamer selection can be made using different systems, but the most frequently used one is currently the yeast two-hybrici system
- an antibody can be, for example, polyclonal or monoclonal
- antibody also comprises derivatives thereof which still retain the binding specificity Techniques for the production of antibodies are well known in the art and described, e g in Harlow and Lane “Antibodies, A Laboratory Manual”, Cold Spring Harbor Laboratory Press, 1988 and Harlow and Lane “Using Antibodies A Laboratory Manual” Cold Spring Harbor Laboratory Press, 1999
- antibody fragments applicable as test compounds include Fab fragments, F ⁇ ab') 2 , Fv or scFv fragments, see, for example, Harlow and Lane (1988) and (1999), be cit
- a small molecule according to the present invention can be organic or inorganic and has a molecular weight of up to 2000 Daitons, preferably not more than 1000 Daitons, most preferably not more than 800 Daitons
- the detection is carried out using a fluorescence microscope
- a fluorescence microscope is a light microscope used to study properties of organic or inorganic substances using the phenomena of fluorescence and phosphorescence instead of, or in addition to, reflection and absorption
- the specimen is illuminated with ⁇ ight of a specific wavelength (or wavelengths) which is absorbed by the fluorophores, causing them to emit longer wavelengths of ⁇ ight (of a different color than the absorbed light)
- the illumination light is separated from the much weaker emitted fluorescence through the use of an emission filter
- Typical components of a fluorescence microscope are the light source (Xenon or Mercury arc-discharge lamp ⁇ , the excitation filter, the dichroic mirror (or dichromatic beamsplitter), and the emission filter
- the filters and the dichroic mirror are chosen to match the spectral excitation and emission characteristics of the fluorophore used to label the specimen
- Most fluorescence microscopes in use are epi-fluorescence microscopes (i e excitation and observation of the fluorescence are from above (ep
- the compounds identified with the present method may be optimized to ar ⁇ ve at a compound which may be, for example, used in a pharmaceutical composition
- Methods for the optimization of the pharmacological properties of compounds identified in screens are known in the art and comprise methods of modifying a compound identified as a iead compound to achieve (i) modified site of action, spectrum of activity, organ specificity, and/or (n) improved potency, and/or (in) decreased toxicity (improved therapeutic index), and/or ( ⁇ v) decreased side effects, and/or (v) modified onset of therapeutic action, duration of effect, and/or (v ⁇ ) modified pharmacokinetic parameters (resorption, distribution, metabolism and excretion), and/or (v ⁇ ) modified physico- chemical parameters (solubility, hygroscopicity, color, taste, odor, stability, state), and/or (viis) improved general specificity, organ/tissue specificity, and/or ( ⁇ x) optimized application form and route by (1) este ⁇ fication of carboxyl
- the present invention relates to the use of the nucieic acid of the invention the vector of the invention, the host ceil of the invention, the polypeptide of the invention or produced by the method of the invention in biomedical research or drug screening in this regard, the embodiments described for the method of screening for compounds having an effect on the cell cycle according to the invention as described above equally apply to the use according to the invention
- Drug screening makes use of the detection of distinct ceil cycle stages like S phase, G1 phase, G2 phase or mitosis in celSs expressing the polypeptide of the invention after treatment with test compounds which can be drugs
- a compound can be but is not restricted to a compound beionging to the classes of e g nucleic acids, (polypeptides, peptide aptamers, nucleic acid based aptamers, small molecules or antibodies or fragments thereof
- the compound can be any chemical compound
- Biomedical research or drug screening preferably includes the screening/identification of compounds which block the entrance of a cell into S phase or which interfere with the progression of S phase, which can be carried out using e g the method of screening for compounds having an effect on the cell cycle as described above This is of particular interest to identify potential drugs which can inhibit genome replication and therefore have a direct impact in cancer treatment by blocking cellular division
- Exemplary screening methods include contacting cells expressing the polypeptide of the invention with test compounds, e g in a multiwell format (96-weil, 384-well or other formats), foilowed by fluorescent live cell microscopy and fluorescence detection as described above Using live cell imaging and fluorescence detection, the polypeptide of the invention can be visualized in the nucleus of a celi after binding to PCNA Thereby, endogenous PCNA can be detected at characteristic structures derived from replication sites ranging from early replicating genes (early S phase) to late replicating chromatin in late S phase Moreover, from the distribution of the polypeptide of the invention within the nucleus it can be concluded whether the cell is in G1 phase G2 phase or in mitosis
- the distribution and the dynamic pattern of the polypeptide of the invention can be directly correlated to the distribution of the polypeptide of the invention in reference cells not contacted with the test compound
- FIG. 3 Visualisation and detection of S phase with a PCNA-Chromobody. Representative images of cells expressing Chromobody PCNA3p16-GFP are shown (A) Coexpressson of the PCNA-Chromobody with other fluorescent S phase markers (RFP-PCNA 1 HcRed-Ligase) colocalising at DNA replication sites (B) Co ⁇ focal images of cells solely expressing the PCNA- Chromobody visualizing different stages of S phase
- FIG. 4 S phase progression is not inhibited by expression of Chromobody PCNA3616-GFP.
- FIG. 5 Representative images of a time lapse analysis of cells expressing Chromobody PCNA3p16-GFP
- Figure 7 Comparison of the ceEiular mobility of GFP-PCNA versus PCNA-Chromobody with FRAP (fluorescence recovery after photobfeaching, FRAP) analysts.
- VHH Alpaca heavy chain antibodies
- PCNA1 pD5-mRFP (F2H) and PCNA3pC8-GFP (F2H) were identical to sequence PCNA1 p15 (EUSA) and PCNA3pA4-mRFP (F2H) to PCNA3p10 (ELISA) and PCNA3p2 (ELISA)
- PCNA1 pD5-mRFP and PCNA3pC8-GFP did not recognize endogenous PCNA in HeLa cells
- PCNA3pF6-GFP was abfe to bind endogenous PCNA (data not shown) and was, therefore, chosen for further characterization
- PCNA3p16 having the amino acid sequence of SEQ ID NO 1 ⁇ when applied as recombinant VHH-domain
- PCNA3p16-GFP having the ammo acid sequence of SEQ ID NO 10 when used as Chromobody
- PCNA3p16-GFP is located at replication sites in HeLa cells and S phase progression is not impeded
- BrdU incorporates in newly synthesized DNA, thereby labeling sites of active DNA replication n cells (red) After a 30 min chase period it was visible that fluorescent BrdU-pattern and PCNA3p16-GFP-pattern (green) were slightly shifted This observation suggests that endogenous PCNA had progressed to adjacent replication foci indicating that binding of PCNA3p16- GFP to endogenous PCNA did not impede S phase progression (Fig 4 B)
- BrdU labeling was performed in cells transiently overexpressing GFP-PCNA Similar to cells expressing PCNA3p16-GFP S phase progression was not inhibited in cells expressing GFP-PCNA
- overexpression of GFP-PCNA sometimes results in a fluorescent pattern similar to a replication pattern although as indicated by BrdU staining cells were not in S-Phase
- Chromobody to replication foci Chromobody to replication foci.
- Figure 7 A shows representative images of half nuclear FRAPs of HeLa cells either expressing Chromobody PCNA3p16-GFP (PCNA-cb-GFP) or GFP-PCNA Statistical evaluation of FRAP measurements of several cells showed a significant faster recovery rate ( ⁇ 35 sec ) of Chromobody PCNA3p16-GFP compared to GFP-PCNA
- This demonstrates a transient interaction between PCNA- Chromobody and endogenous PCNA in living cells The transient nature of this interaction is most probably the reason that the function of endogenous PCNA is not disturbed or impeded by the binding of PCNA3p16-GFP
- the binding is ⁇ tiil specific enough to enable the visualization of the endogenous PCNA protein at replication foci throughout S phase inside hvmg cells ( Figure 7 B)
- HeLa cell lines stably expressing PCNA3p16-GFP were generated HeLa cells were seeded at very low density (1 % confluence) and transfected with PCNA3p16-GFP expression vectors 2 days after transfectton, cells were cultured in medium containing G418 to select transfected cells After several days ceils expressing PCNA3p16-GFP were observed to form colonies Colonies with homogenous and appropriate expression levels for the detection of replication patterns ( ⁇ e expression levels enabling both for the detection of PCNA3p16-GFP and for a spatial resolution of focal structures) were chosen for further subclomng Finally, three clones 5 1 , 9 1 and 9 6 were selected (Fig. 8 A)
- Example 7 Conservative substitutions in the framework regions do not impede functionality of the polypeptide.
- HeLa cells were seeded on coverslips and transiently transfected with expression vectors coding for a variant PCNA-Chromobody of SEQ ID NO 16 and fused to GFP or a variant PCNA- Chromobody of SEQ ID NO 18 and fused to GFP 24 h after transfection cells were fixed in 4% PFA for 15 minutes at room temperature, permeabsiized with 0,2% T ⁇ ton-X-100 in PBS and the DNA was counterstained with DAPI Slides were mounted in VectaShield and further analyzed by epifluorescence microscopy with appropriate filtersets for DAPI (D360_40X / HQ460_40M) and GFP (HQ480_40X / HQ535_50M) and an 63X/1 3 NA Plan Apo oil objective
- Chromobodies recognize PCNA and localize correctly at replication foci in cells (see Figure 10) From this it can be concluded that conservative mutations tn the framework do not interfere with the antigen binding capacity of the corresponding chromobodies
- Example 8 Drug screening using the nucleic acid of the invention on the ⁇ xampie of the mitotic inducer coicemide HeLa cell-lines stably expressing PCNA3p16-GFP (clone 9 6, amino acid sequence of SEQ ID NO 2 fused to GFP) were seeded at 50% confluency in live cell imaging chamber (ibsdi ⁇ -Slide 8 well) After incubation over night, normal growth medium (DMEM + 10% FCS) was replaced with CO 2 adapted live-cell medium (DMEM without phenol red + 10% FCS + 25mM Hepes Buffer) Subsequently the imaging slide was placed on an INCeII Analyzer 1000 (GE Healthcare) high content microscope equipped with an environmental control chamber, adjusted to 37°C and 5% CO 2 Images were acquired with an 20X/0 45 NA air objective using the appropriate filterset for the fluorescent protein GFP (HQ480_40X / HQ535__50M) and an exposure time of 750 ms For the live cell imaging 15 fields of view per well
- Coicemid was added directly to the medium of one well, control cells were left untreated (figure 11A)
- Coicemid is a mitotic inhibitor drug such as Colchicine, which depolyme ⁇ ses microtubules and limits microtubule formation (inactivates spindSe fibre formation), thus arresting cel ⁇ s in metaphase
- the acquired data set was analyzed with the automated image evaluation and quantification program INCeI! workstation For that a specialized protocol was designed and developed to identify the cell, the nudes and to distinguish between cells in interphase and metaphase The amount of mitotic ceils per time point was counted and evaluated The plotted graph in figure 11 B shows the expected increase of mitotic cells in the coicemid treated eel! population
- Lukyanov K.A A F Fradkov, N G Gurskaya, M V Matz, Y A Labas, A P Savitsky, M L Markelov, A G Zaraisky, X Zhao, Y Fang, W Tan, and S A Lukyanov 2000 Natural animal coloration can Be determined by a nonfluorescent green fluorescent protein homolog J Biol Chem 275 25879-82
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WO2013126674A1 (en) * | 2012-02-23 | 2013-08-29 | Anthrogenesis Corporation | Identification of antitumor compounds using placenta |
WO2014007634A1 (en) | 2012-07-06 | 2014-01-09 | Ihc Holland Ie B.V. | Cutter head for removing material from a water bed |
JP2015504313A (en) * | 2011-12-02 | 2015-02-12 | フェイト セラピューティクス,インコーポレイテッド | Enhanced stem cell composition |
WO2016077526A1 (en) | 2014-11-12 | 2016-05-19 | Siamab Therapeutics, Inc. | Glycan-interacting compounds and methods of use |
WO2017083582A1 (en) | 2015-11-12 | 2017-05-18 | Siamab Therapeutics, Inc. | Glycan-interacting compounds and methods of use |
US9879087B2 (en) | 2014-11-12 | 2018-01-30 | Siamab Therapeutics, Inc. | Glycan-interacting compounds and methods of use |
US11253609B2 (en) | 2017-03-03 | 2022-02-22 | Seagen Inc. | Glycan-interacting compounds and methods of use |
US11401330B2 (en) | 2016-11-17 | 2022-08-02 | Seagen Inc. | Glycan-interacting compounds and methods of use |
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KR102073748B1 (en) * | 2013-01-31 | 2020-02-05 | 한미약품 주식회사 | Recombinant yeast transformant and process for preparing immunoglobulin Fc fragment employing the same |
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