EP1313852A2 - Domaines peptidiques liant l'adn et procede de preparation de tels domaines - Google Patents

Domaines peptidiques liant l'adn et procede de preparation de tels domaines

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Publication number
EP1313852A2
EP1313852A2 EP01971927A EP01971927A EP1313852A2 EP 1313852 A2 EP1313852 A2 EP 1313852A2 EP 01971927 A EP01971927 A EP 01971927A EP 01971927 A EP01971927 A EP 01971927A EP 1313852 A2 EP1313852 A2 EP 1313852A2
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EP
European Patent Office
Prior art keywords
amino acid
biomorphic
seq
transcription
peptides
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EP01971927A
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German (de)
English (en)
Inventor
Andreas Kappel
Norbert Windhab
Thomas Wagner
Stefan Kienle
Karsten Kuhn
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Xzillion GmbH and Co KG
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Xzillion GmbH and Co KG
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Publication of EP1313852A2 publication Critical patent/EP1313852A2/fr
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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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1082—Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity

Definitions

  • DNA binding peptide domains and a method for providing such domains.
  • the present invention relates to easily synthetically accessible peptide domains that can specifically recognize and bind nucleic acid sequences, and to a method for finding and providing specifically DNA-binding peptide domains, and biomorphic factors derived therefrom, in particular transcription factors and repressors.
  • RNA Transcription of DNA into RNA is the first step in gene expression.
  • the strength of the transcription of a gene and thus the amount of RNA formed is determined by regulatory promoter elements and enhancer elements associated with the genes, to which transcription factors bind.
  • these proteins bind sequence-specifically to short DNA sections in the regulatory elements of the genes.
  • transcription factors contain one or more activator or repressor domains. Activator domains increase the transcription of the gene belonging to the regulatory element bound by the transcription factor, while repressor domains weaken the transcription of the gene. Both are done by recruiting additional proteins through the activator or repressor domains via protein-protein interactions.
  • the DNA sequence of the regulatory elements of a gene and the protein domains that bind specifically to this sequence therefore act as complementary addresses which, when interacting, locate activator and repressor domains, which ultimately determine the transcriptional strength of a gene.
  • Another method involves introducing relatively large amounts of short double-stranded DNA molecules into cells that contain a binding site for a transcription factor that is responsible for the deregulated transcription of the respective gene.
  • the selected transcription factor is competitively intercepted and can therefore no longer bind to the binding sites in the regulatory elements of its target genes, as a result of which the transcription of these genes is indirectly inhibited (overview in Suda et al., Endocr. Rev., 1999 , 20, 345-357; S. Yla-Hertttuala and JF Martin, The Lancet 355, 213-222, 2000).
  • a relatively new method for regulating transcription is represented by synthetic transcription factors from the class of zinc finger proteins from Cys 2 -His 2 (DJ. Segall, B. Dreier, RRBeerli, C, F. Barbas III, Proc. Natl. Acad. Sei. USA , 1999, 96, 2758-2763). These factors bind to the large groove of the DNA by means of a finger-like structure in which a short ⁇ -helix and an antiparallel ⁇ -sheet are complexed together by a zinc atom (D. Rhodes, A. Klug, Sei. Am., 1993, 268 , 32-39).
  • the DNA binding activity of the zinc finger proteins has a modular structure.
  • multifinger proteins are usually encoded in a phage library, which is why the members of the library that bind to a specific DNA sequence of the target gene can be isolated using the phage display methodology.
  • the cDNA of the DNA-binding polypeptide is then fused with the cDNA of an activator or repressor domain; the resulting fusion protein thus represents a specific regulator of the target gene (overview of the methodology in DJ. Segal and CF Barbas III, Curr. Opin Chem. Biol., 2000, 4, 34-39; A. Klug, J. Mol. Biol. , 1999, 293, 215-218).
  • the task is to provide short peptide biomorphic factors that can specifically recognize and bind nucleic acid sequences and to provide a method for the discovery of such biomorphic factors.
  • Any two peptides are preferably selected from this group, which form a homo- or heterodimeric molecule or a mixture of homo- and heterodimeric molecules via a suitable linker.
  • Structural variants are understood to be amino acid sequences which have at least a homology of 75%, preferably a homology of more than 90%, to the Seq. Have ID No.1. Functional variants are preferred which can enter into a sequence-specific DNA binding.
  • linkers All compounds which dimerize two peptides from the group with the Seq are suitable as linkers. ID No. 1 or their structural variants in solution.
  • the linkers can be free organic or inorganic substances that can be added to the peptides according to the invention. Examples of this are e.g. B. complexing agents that can form coordinative bonds with the free amino or carboxy termini of peptides.
  • the linkers and peptides can also be covalently linked by chemical modification of at least one of the peptides.
  • Peptide linkers can also be fused directly to at least one of the peptides. Fusion proteins are preferred which, in addition to a DNA-binding domain according to Seq ID No. 1 or a structural variant have a leucine zipper amino acid domain.
  • Seq. ID No. 2 exemplifies the amino acid sequence of such a fusion protein.
  • the peptides according to the invention can be prepared synthetically using methods known to the person skilled in the art (for example after Merryfield synthesis), the peptides can also be obtained by molecular biological methods, such as expression in cell culture or in fermenters.
  • the short dimers the peptides with Seq. ID No. 1 or its variants contain specific DNA sequences and can therefore regulate the activity of genes in living cells. Due to the shortness of the amino acid sequence, the peptides are easily accessible synthetically, and due to the shortness of the peptides, the transport through cell membranes is facilitated.
  • the high stability of the conformation of the peptides according to the invention ensures that their function, that is to say the ability to bind DNA in a sequence-specific manner, is retained during transport through cell membranes. Due to the different structure of the peptides compared to other artificial transcription factors, certain DNA sequence motifs can also be better bound.
  • the dimers according to the invention are suitable as a module for the construction of artificial activating or repressing transcription factors.
  • the DNA binding domains are fused with known nuclear transport signal domains, transcription activation domains or transcription inhibition domains such as, for example, the core localization signal of the SV40 T antigen, the transcription activator domain of the viral VP16 protein, or the KRAB repressor domain.
  • the artificial biomorphic factors generated in this way can be used to control transcription and thus also to regulate the expression of genes.
  • the artificial biomorphic factors according to the invention thus enable the investigation of phenotypic effects of expression changes of individual genes in vivo. Above all, the use of such biomorphic factors to combat diseases that are based on misregulated gene transcription, such as. B. cancer, inflammatory reactions or addictions is of great interest.
  • the biomorphic factors are used as biopharmaceuticals, their easy physiological degradability is also advantageous.
  • the present invention therefore furthermore also relates to a medicament which contains the peptide monomers or dimers according to the invention and, if appropriate, suitable additives or auxiliaries, and to a process for the preparation of a medicament for the treatment of diseases which result from misregulated or from the expression of mutated genes , in which a peptide monomer or dimer according to the invention is formulated with pharmaceutically acceptable additives and / or auxiliaries.
  • the invention further relates to the peptides according to the invention according to Seq ID No. 1 or DNA sequences encoding their structural variants (Seq. ID No. 3) or nucleic acids comprising these sequences.
  • Structural variants are understood to be nucleic acids with a different sequence, the proteins according to Seq ID No. 1 or code their structural variants. Preference is given to nucleic acids which encode functional variants of these proteins.
  • DNA sequences which code for fusion proteins from the DNA-binding domains according to the invention and a leucine zipper domain are preferred.
  • nucleic acids according to the invention can encoding areas such. B. for Kemtspragsignaldomainen (nuclear localization signals), transcription activation domains (z. B. from transcription factors) or transcription inhibition domains.
  • the nucleic acids according to the invention can for example be chemically based on the sequences disclosed in SEQ ID No 3 or 4 or on the basis of the peptide sequences disclosed in SEQ ID No 1 or 2 or their structural variants using the genetic code z.
  • B. can be synthesized by the phosphotriester method (see e.g. Uhlman, E. & Peyman, A. (1990) Chemical Reviews, 90, 543, No. 4).
  • deoxyribonucleic acids according to the invention can be used for gene therapy purposes but also for the investigation of phenotypic effects by changing the expression of individual genes in vivo. For this, DNA fragments must be introduced into suitable expression vectors.
  • pro- or eukaryotic expression vectors can be used as vectors.
  • virus vectors preferably adenovirus vectors, in particular replication-deficient adenovirus vectors, or adeno-associated virus vectors, for example an adeno-associated virus vector which consists exclusively of two inserted terminal repeat sequences (ITR).
  • adenovirus vectors are described, for example, in McGrory, WJ. et al. (1988) Virol. 163, 614; Gluzman, Y. et al. (1982) in ⁇ ukaryotic Viral Vectors "(Gluzman, Y. ed.) 187, Cold Spring Harbor Press, Cold Spring Habor, New York; Chroboczek, J. et al. (1992) Virol. 186, 280; Karlsson, S. et al. (1986) EMBO J .. 5, 2377 or WO95 / 00655.
  • Suitable adeno-associated virus vectors are described, for example, in Muzyczka, N. (1992) Curr. Top. Microbiol. Immunol. 158, 97; WO95 / 23867; Samulski, RJ. (1989) J. Virol, 63, 3822; WO95 / 23867; Chiorini, J.A. et al. (1995) Human Gene Therapy 6, 1531 or Kotin, R.M. (1994) Human Gene Therapy 5, 793.
  • Vectors with gene therapy effects can also be obtained by complexing the nucleic acid according to the invention with liposomes.
  • Lipid mixtures such as those from Feigner, P.L. et al. (1987) Proc. Natl. Acad. Sei, USA 84, 7413; Behr, J.P. et al. (1989) Proc. Natl. Acad. Be. USA 86, 6982; Feigner, J.H. et al. (1994) J. Biol. Chem. 269, 2550 or Gao, X. & Huang, L. (1991) Biochim. Biophys. Acta 1189, 195.
  • the DNA is ionically bound to the surface of the liposomes in such a ratio that a positive net charge remains and the DNA is completely complexed by the liposomes.
  • nucleic acids according to the invention are therefore in an expression vector, preferably in an expression vector which is suitable for gene therapy use or for the production of transgenic microorganisms, plants or animals.
  • the present invention therefore furthermore also relates to a medicament which, using the nucleic acids according to the invention, is suitable, if appropriate, using further additives or auxiliaries for gene therapy use in diseases which are due to misregulated or to the expression of mutated genes.
  • a medicament is suitable which contains the nucleic acids according to the invention in naked form or in the form of one of the above contains described gene therapy vectors or in complexed form with liposomes.
  • Suitable additives and / or auxiliary substances are e.g. a physiological saline, stabilizers, proteinase inhibitors, nuclease inhibitors etc.
  • Another object of the invention is a library which contains deoxyribonucleic acids, the sequences according to Seq. ID No. 3 or have a structural variant thereof and the coding regions of which contain a transcription activation domain (AD) and optionally a core transport sequence domain (NLS) in the open reading frame. It is also preferred if the DNAs additionally contain a linker-coding region, particularly preferably a leucine zipper (FIG. 1).
  • the components of the library encoding peptide monomers form, in solution, preferably under physiological conditions, homo- and / or heterodimeric biomorphic transcription factors.
  • An example is the structural structure of the members of a library containing deoxyribonucleic acids, comprising regions coding for NLS and AD, in Seq. ID No 5 and Fig. 1 shown.
  • the library contains DNA sequences which code for the individual monomers of the biomorphic transcription factors and whose peptide monomers after expression in a transformed expression system, such as. B. in e. coli or in yeast, via suitable linker dimerization.
  • pro- and / or eukaryotic expression vectors can be used as vectors.
  • the expression vectors also contain suitable regulatory sequences for the host cell, such as e.g. B. the trp promoter for expression in e. coli or the ADH-2 promoter for expression in yeast, the baculovirus polyhedrin promoter for expression in insect cells.
  • Libraries are preferred which contain all possible cDNA sequences according to Seq. ID No 3 or a structural variant contained one or more times.
  • the DNA sequences of the library can be present and stored in sequential form, in the form of a suitable vector or in the form of a cellular expression system transformed with these vectors.
  • the invention further encompasses the biomorphic transcription factors expressible with the aid of these libraries.
  • biomorphic transcription factors expressible with the aid of these libraries.
  • pure peptide libraries can also be obtained from biomorphic transcription factors.
  • the artificial synthesis of biomorphic transcription factors to create a peptide library can also be carried out.
  • An example is the structural structure of the members of a library containing peptides in Seq. ID No 6 shown.
  • Another object is a method for finding peptidic biomorphic factors that can specifically recognize and bind nucleic acid sequences.
  • a cellular expression system is chosen in which an essential gene is defective.
  • the cellular expression system is transformed with a corresponding wild-type gene that is controlled by a promoter that allows basal transcription (insertion element).
  • the transforming construct contains a DNA sequence which is to be tested for sequence-specific binding biomorphic factors (response element).
  • Regulatory DNA sequences such as promoters or operators are of particular interest as the response element.
  • gene-specific DNA sequences of the coding region of a gene in particular specific DNA sequences of a mutated region of a gene, are also preferred objects of investigation. All lethal defect mutants which can be cultivated via an inhibitable basal transcription of an introduced wild-type gene or via a directly lethally inhibitable gene product can be used as the cellular expression system. Easily cultivable microorganisms, such as. B. e. coli or yeasts are used, but cell cultures from higher organisms can also be used as an expression system.
  • known HIS - yeast cells can be used as an expression system.
  • the yeast cells are transformed with a construct that a DNA sequence with a wild-type binding site, e.g. B. contains for transcription factors, enhancers or repressors in one or more versions.
  • the double-stranded sequences to be tested for binding biomorphic factors can be inserted into a vector containing a wild-type HIS gene, such as the pHISi vector (Clontech, Heidelberg).
  • the HIS gene of the pHISi vector is under the control of a minimal promoter.
  • the method according to the invention for finding sequence-specific DNA-binding biomorphic transcription factors and thus suitable binding domains comprises the following method steps:
  • Promoters and a response element with a bank of expression vectors containing the invention
  • Binding site (Fig. 2A) highlighted in gray) contains three (3xE2Fwt sequence, Seq. ID
  • each of the single-stranded 10 ⁇ M oligonucleotides 3xE2F wtsense Seq. ID No 7 and 3xE2F wtas Seq. ID No 8 were in 100 ⁇ l 10mM Tris-HCI pH7.9, 50mM NaCI,
  • the 3xE2Fwt sequence is cloned into the Kpnl and Sacl restriction sites of the pGL-2 vector (Promega, Madison, Wl USA).
  • 3 ⁇ g pGL-2 with 20u Kpnl and Sacl each were completely digested and purified using an agarose gel.
  • the DNA was freed from agarose residues using the QiaQuick Gel Extraction Kit (Qiagen / Hilden) and taken up in 50 ⁇ l water.
  • 10 ⁇ l of the purified vector were ligated with 1 ⁇ l of the double-stranded oligo using the T4 DNA ligase in a 20 ⁇ l mixture for 2 h at 25 ° C.
  • the E.coli k12 strain "Goldstar” (Stratagene, La Jolla, San Diego, USA) was shaken and grown overnight at 37 ° C. and 200 RPM in LB medium with 100 ⁇ g / ml ampicillin. The next morning, 1 ml the bacterial culture was inoculated into 200 ml of fresh medium and shaken to an optical density of 0.565 at 595 nm at 37 ° C. and 200 RPM, the culture was then cooled to 4 ° C.
  • the bacteria were mixed with 1 ml LB medium and incubated with shaking at 37 ° C. for one hour. The mixture was then spread on LB agar plates with 100 ⁇ g / ml ampicillin and incubated at 37 ° C. overnight. Individual clones were isolated and grown in 3 ml LB medium with 100 ⁇ g / ml ampicillin at 37 ° C. overnight. The plasmid DNA was isolated and purified from the bacteria using the QIAprep Spin Miniprep Kit (Qiagen / Hilden) according to the manufacturer's instructions.
  • Positive clones were identified by PCR as follows: 1 ⁇ l DNA, 0.5 ⁇ l corresponding to 5U Taq polymerase (Promega, Madison, USA), 5 ⁇ l 10 ⁇ reaction buffer (Promega, Madison, USA), 0.4 ⁇ l deoxynucleotide triphosphate (jelO ⁇ M), 4 ⁇ l 12.5mM MgCI 2 , as well as 0.3 ⁇ l of 100 ⁇ M oligonucleotides and distilled water ad 50 ⁇ l were heated to 94 ° C for 3min. This was followed by 30 cycles at 94 ° C. for 20 seconds, 50 ° C. for 20 seconds, and 72 ° C. for 45 seconds, followed by an incubation at 72 ° C. for 5 minutes.
  • PCR products were separated by agarose gel electrophoresis and thus their size was determined.
  • Bacterial colonies whose plasmid DNA had a PCR product of the expected size were shaken in 50 ml LB medium with 100 ⁇ g / ml ampicilin for 13 hours at 37 ° C. and 250 RPM.
  • the plasmid DNA was then isolated from the bacteria and purified using the Qiagen Midi Prep Kit (Qiagen / Hilden).
  • the DNA was resuspended in distilled water.
  • the 3xE2Fwt sequence thus amplified was then isolated from the vector. For this purpose, 3 ⁇ g of the plasmid with 20U Xmal and Mlul were digested completely for 3 hours.
  • the mixture was then separated on an agarose gel, and the 3xE2Fwt insertion sequence migrating at 68 bp was cut out with a scalpel.
  • 3 ⁇ g pHISi (Clontech, Heidelberg), each with 20U Xmal and Mlul, were completely digested for 3 hours and cleaned using an agarose gel.
  • the DNA samples were then freed of agarose using the QiaQuick Gel Extraction Kit (Qiagen / Hilden) according to the manufacturer's instructions and taken up in 50 ⁇ l water.
  • the culture was then cooled to 4 ° C. and centrifuged at 2500 ⁇ g, the supernatant was discarded and the pelleted bacteria were removed in 7.5 ml LB medium with 10% (w / v) polyethylene glycol-6000, 5% dimethyl sulfoxide, 10mM MgSO 4 , 10mM (Promega, Madison, USA), pH 6.8, incubated for one hour on ice, snap frozen in liquid nitrogen and stored at -80 ° C.
  • 10 ⁇ l of the ligation mixture was taken up in 100 ⁇ l 100 mM KCI, 30 mM CaCl 2 , 50 mM MgCl 2 and incubated with 100 ⁇ l of the thawed bacteria for 20 min on ice
  • the bacteria were mixed with 1 ml LB medium and incubated with shaking at 37 ° C. for one hour.
  • the mixture was then spread on LB agar plates with 100 ⁇ g / ml ampicillin and incubated at 37 ° C. overnight.
  • Individual clones were isolated and grown in 3 ml LB medium with 100 ⁇ g / ml ampicillin at 37 ° C. overnight.
  • the plasmid DNA was isolated and purified from the bacteria using the QIAprep Spin Miniprep Kit (Qiagen / Hilden) according to the manufacturer's instructions. Positive clones were identified by digestion with Xmal and Mlul. The corresponding bacteria were shaken in 50 ml LB medium with 100 ⁇ g / ml ampicilin for 13 hours at 37 ° C. and 250 RPM. The plasmid DNA was then isolated from the bacteria and purified using the Qiagen Midi Prep Kit (Qiagen / Hilden). The DNA was resuspended in distilled water. The resulting plasmid is called pHISi3xE2Fwt (Seq. ID. No. 9).
  • the HIS3 gene of the vector is placed under the control of the insertion element in addition to the basal HIS3 promoter present in the vector.
  • the yeast strain YM 4271 (Clontech, Heidelberg) is treated with the pHISi-3xE2Fwt vector thus prepared according to the manufacturer's instructions (Yeast Protocols Handbook, Clontech, Heidelberg pp.
  • This culture was incubated with shaking at 30 ° C until it had an optical density at 600 nm of 0.4-0.6. The culture was then centrifuged at 1000xg for 5 minutes and the cells were taken up in 1.5 ml of 100 mM lithium acetate pH 7.5, 10 mM Tris-HCl pH 7.5, 1 mM EDTA.
  • 100 ⁇ l of the cells, 50 ⁇ l of the linearized plasmid and 100 ⁇ g of heat-denatured salmon sperm DNA were mixed with 600 ⁇ l of a PEG / LiAc solution (100mM lithium acetate pH 7.5, 10mM Tris-HCl pH7.5, 1mM EDTA, 40% (w / v) polyethylene glycol) 4000) and shaken at 30 ° C for 30 min. After the addition of 70 ⁇ l dimethyl sulfoxide, the mixture was incubated at 42 ° C. for 15 min.
  • a PEG / LiAc solution 100mM lithium acetate pH 7.5, 10mM Tris-HCl pH7.5, 1mM EDTA, 40% (w / v) polyethylene glycol
  • the cells were then centrifuged off, washed in 1 ml of 10 mM Tris-HCl pH7.5, 1 mM EDTA and completely plated onto a minimal medium agar plate without histidine addition, on which 12 colonies had grown after 7 days at 30 ° C.
  • HIS3 The low basal expression of HIS3 allows the selection of transformed YM 4271 yeasts which have inserted the insertion element into their chromosomal DNA by dislodging the cell cultures on SD agar plates with histidine-free medium. The YM 4271 yeast strains growing under these conditions are isolated.
  • the increasing amounts of the HIS3 antagonist 3-aminotriazole (3-AT) contained OmM, 0.5mM, 1mM, 1.5mM, 2mM, 3mM, 6mM, 9mM , 12mM, 15mM, 18mM, 30mM, 45mM; one clone was already evident at 0.5mM and completely suppressed at 2mM; this and another clone no longer grew at 3mM, while the 10 others were only inhibited in growth at> 30mM.
  • yeast3xE2Fwt The growth of one of these colonies, designated as yeast3xE2Fwt, was already completely suppressed at a concentration of 2mM 3-AT (FIG. 2D) without antagonists (FIG. 2C)). This colony was isolated and used for further investigations.
  • the selected strain yeast3xE2Fwt has an advantageous very low basal expression level of HIS3, which is competitively inhibited even by small amounts of 3-AT.
  • the strain is called YM 4271 3xE2Fwt
  • 3 ⁇ g pGADD424 (Clontech / Heidelberg, Seq. ID No 11) were completely digested with 20u EcoRl and BamHI each and purified using an agarose gel. 1 ⁇ g of the double-stranded peptide domain encoding oligonucleotide Seq ID No 10 was synthesized and digested for 3 h with 10 ⁇ EcoRI and Bam HI in each case and purified using an agarose gel. Subsequently, the DNA was freed from agarose residues using the QiaQuick Gel Extraction Kit (Qiagen / Hilden) and taken up in 50 ⁇ l water.
  • QiaQuick Gel Extraction Kit Qiagen / Hilden
  • 3 ⁇ l of the vector were ligated with 0.1 ⁇ l, 0.3 ⁇ l and 1 ⁇ l of the oligonucleotide for 3 h at room temperature in 20 ⁇ l batches using the T4-DNA ligase. The batches were then shaken with 200 ⁇ l each of n-butanol and centrifuged for 10 minutes at 13000 rpm in a table centrifuge. The supernatants were discarded and the DNA was removed from butanol residues by evaporation in vacuo and taken up in 10 ⁇ l water each. For the transformation, 500 ml of an exponentially growing bacterial culture of the E.
  • coli K12 strain TOP10 was centrifuged for 10 min at 4000xg, resuspended in 500 ml of cold water and centrifuged again. This procedure was repeated twice. The bacteria were then taken up in 7.5 ml of 10% glycerol (v / v) and snap-frozen as 40 ⁇ l aliquots in liquid nitrogen. For the actual transformation, 5 ⁇ l each of the purified ligation batches described above were mixed with an aliquot of the bacteria thawed on ice and transferred to a cold electroporation cuvette.
  • the bacteria were made for shaken for 1 hour in 1 ml LB medium at 37 ° C and 250 RPM and then incubated on LB agar plates with 100 ⁇ g / ml ampicilin at 37 ° C overnight. The following day, the clones of the densely overgrown plates were scraped off and shaken in 250 ml LB medium with 100 ⁇ g / ml ampicilin for 3 hours at 37 ° C. and 250 RPM. The plasmid DNA was then isolated from the bacteria and purified using the Qiagen Maxi prep kit (Qiagen / Hilden). The DNA was resuspended in distilled water and had a concentration of 1.3 ⁇ g / ⁇ l.
  • the generated cDNA library is transformed into the yeast strain yeast3xE2Fwt according to the manufacturer (Yeast protocols handbook, Clontech, Heidelberg) as follows:
  • the cells were then centrifuged, washed in 10 mM Tris-HCl pH7.5, 1 mM EDTA and completely on 6 minimal medium agar plates without the addition of JHistidine and leucine and 2mM 3- Aminotriazole plated on which 153 colonies had grown after 7 days at 30 ° C.
  • yeast strains which contain both the insertion element and a library component including the pGAD424 vector Leu2 marker is carried out simply by plating on SD agar plates without histidine and leucine.
  • the yeast colonies growing under the given selection conditions are transformed with library components which code for expressible biomorphic transcription factors, the action of which is specifically directed towards the HIS3 gene and the E2F binding site, the only multiply occurring sequence of the HIS3 promoter (the HIS3 expression can be regulate only by factors that bind to multiple repetitions of the sequence, since only the binding of several transcription factors leads to a cooperative and thus steep increase in transcription initiation.
  • These colonies therefore have library members whose expression products specifically bind to the trimerized E2F binding site of the HIS3 gene construct.
  • the plasmids were isolated from 32 of the yeast cell cultures, transformed into E. coli and propagated (Yeast protocols handbook, Clontech, Heidelberg).
  • yeast colonies were shaken in 0.5 ml SD minimal medium without addition of histidine and leucine at 30 ° C. overnight. The next day the cells Pellettiert by centrifugation and removed so much from the medium that about 50 ⁇ l remained. In this medium residue, the cells were resuspended by vortexing and a spatula tip of the cell wall-destroying enzyme lyticase was added. The cells were incubated for one hour at 37 ° C. while shaking, and 10 ⁇ l of a 20% sodium dodecyl sulfate solution were then added. For lysing, the cells were vortexed for 1 min, briefly frozen at -20 ° C. and thawed quickly.
  • the cell debris was separated by centrifugation at 14,000 rpm, and the plasmid DNA present in the supernatant was freed of impurities using the QiaQuick PCR Purification Kit and taken up in 50 ⁇ l water.
  • the plasmid DNA was then transformed into bacteria and expanded in order to obtain larger amounts of the plasmids.
  • the E.coli k12 strain “Goldstar” (Stratagene, La Jolla, San Diego, USA) was shaken and grown overnight at 37 ° C. and 200 RPM in LB medium with 100 ⁇ g / ml ampicillin.
  • the isolated plasmid DNA was taken up in 100 ⁇ l 100 mM KCI, 30 mM CaCl 2 , 50 mM MgCI 2 and incubated with 100 ⁇ l of the thawed bacteria for 20 min on ice, after a 10-minute period incubation at room temperature, the bacteria were mixed with 1 ml LB medium and incubated at 37 ° C. with shaking for one hour. The mixture was then spread on LB agar plates with 100 ⁇ g / ml ampicillin and incubated at 37 ° C. overnight. A single clone was grown from each transformation in 3 ml LB medium overnight at 37 ° C. The plasmids were then prepared and purified using the Qiagen Tip20 kit according to the manufacturer's instructions (Qiagen, Hilden).
  • the domains have amino acids with very similar properties at the positions crucial for sequence-specific binding, e.g. B. valine and isoleucine (shaded).
  • a consensus sequence (Seq. ID No. 13) can be derived from the individual sequences.
  • a consensus sequence of the recognition amino acids (item X-1: G or C, item X-2: V, item X-3: V, item X-4: G) of the novel peptides can be derived from the comparison of the amino acid sequence of the binders which, however, did not appear as a separate sequence among the analyzed library members. However, this can be justified by the fact that with an estimated library transformation efficiency of approximately 200,000 transformation events and a ratio vector (with insert vector (without insert) of 1: 5, only approximately 40,000 different library members were transformed. With a complexity of the library of 104,000 coding Members were therefore transformed into yeast with a probability of 0.38.

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  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Toxicology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Virology (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne des domaines peptidiques aisément accessibles de manière synthétique, qui reconnaissent spécifiquement et lient des séquences d'acide nucléique. L'invention concerne en outre un procédé permettant de détecter et de préparer des domaines peptidiques liant l'ADN de manière spécifique, ainsi que des facteurs biomorphes qui en sont dérivés, notamment des facteurs de transcription et des répresseurs.
EP01971927A 2000-08-22 2001-08-20 Domaines peptidiques liant l'adn et procede de preparation de tels domaines Withdrawn EP1313852A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10041126 2000-08-22
DE10041126A DE10041126A1 (de) 2000-08-22 2000-08-22 DNA-bindende Peptiddomänen und ein Verfahren zur Bereitstellung solcher Domänen
PCT/EP2001/009565 WO2002016424A2 (fr) 2000-08-22 2001-08-20 Domaines peptidiques liant l'adn et procede de preparation de tels domaines

Publications (1)

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EP1313852A2 true EP1313852A2 (fr) 2003-05-28

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EP01971927A Withdrawn EP1313852A2 (fr) 2000-08-22 2001-08-20 Domaines peptidiques liant l'adn et procede de preparation de tels domaines

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US (1) US20040072180A1 (fr)
EP (1) EP1313852A2 (fr)
AU (1) AU2001291774A1 (fr)
CA (1) CA2420251A1 (fr)
DE (1) DE10041126A1 (fr)
WO (1) WO2002016424A2 (fr)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8999932B2 (en) 2009-07-29 2015-04-07 Kai Pharmaceuticals, Inc. Therapeutic agents for reducing parathyroid hormone levels
EP3031820A1 (fr) 2014-12-08 2016-06-15 Life Science Inkubator Particules viroides (VLP) comportant un peptide de ciblage
EP3031821A1 (fr) * 2014-12-08 2016-06-15 Life Science Inkubator Particules viroïdes (VLP) issues de polyomavirus comportant une protéine de fusion

Family Cites Families (2)

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Publication number Priority date Publication date Assignee Title
US5783384A (en) * 1992-01-13 1998-07-21 President And Fellows Of Harvard College Selection of binding-molecules
US5869250A (en) * 1996-12-02 1999-02-09 The University Of North Carolina At Chapel Hill Method for the identification of peptides that recognize specific DNA sequences

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0216424A2 *

Also Published As

Publication number Publication date
WO2002016424A3 (fr) 2002-05-16
US20040072180A1 (en) 2004-04-15
CA2420251A1 (fr) 2002-02-28
DE10041126A1 (de) 2002-04-04
AU2001291774A1 (en) 2002-03-04
WO2002016424A2 (fr) 2002-02-28

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