WO1997045538A1 - Nouvelles matrices structurelles proteiques synthetiques pour la generation, le criblage et l'evolution de surfaces moleculaires fonctionnelles - Google Patents

Nouvelles matrices structurelles proteiques synthetiques pour la generation, le criblage et l'evolution de surfaces moleculaires fonctionnelles Download PDF

Info

Publication number
WO1997045538A1
WO1997045538A1 PCT/EP1997/002840 EP9702840W WO9745538A1 WO 1997045538 A1 WO1997045538 A1 WO 1997045538A1 EP 9702840 W EP9702840 W EP 9702840W WO 9745538 A1 WO9745538 A1 WO 9745538A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
sequence
domain
seq
protein
Prior art date
Application number
PCT/EP1997/002840
Other languages
English (en)
Inventor
Boris Steipe
Heike Bruhn
Martin Funk
Thomas Henkel
Original Assignee
Medigene Ag
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Medigene Ag filed Critical Medigene Ag
Priority to EP97927094A priority Critical patent/EP0912728A1/fr
Priority to JP09541632A priority patent/JP2000511050A/ja
Publication of WO1997045538A1 publication Critical patent/WO1997045538A1/fr

Links

Classifications

    • 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
    • C12N15/1044—Preparation or screening of libraries displayed on scaffold proteins
    • 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
    • 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
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/42—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a HA(hemagglutinin)-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor

Definitions

  • Novel synthetic protein structural templates for the generation, screening and evolution of functional molecular surfaces are novel synthetic protein structural templates for the generation, screening and evolution of functional molecular surfaces .
  • the present invention relates generally to protein molecules with novel binding or catalytic properties. More specifically, the invention relates to tne production of libraries of peptide sequences m the framework of a structural template derived from Pleckstrm-Homology (PH) domains and the identification of such sequences that posses the desired properties of binding macromolecular or small molecule ligands, including the transition states of chemical reactions. The invention also relates to the provision of small molecules, derived from the so ontamed peptide sequences that posses ligand binding properties comparable to those of the peptides m the context of the structural framework.
  • PH Pleckstrm-Homology
  • Proteins are complex macromolecules that are assembled in the cell as linear heterocopolymers of ammo acids Their precise composition essentially is encoded in the genetic sequence and the protein sequence uniquely and robustly specifies the three-dimensional fold of the protein It is this fold, the spatial organization of molecular shape, hydrophobic and hydrophilic areas and electrostatic fields which is at the basis of the specificity of molecular interactions that ultimately determines the function of the proteins
  • the underlying principle is one of molecular recognition oy complementarity of interacting surfaces Since the shape and nature of protein surfaces is determined by the three- dimensional structure of the protein, which ir. turn is determined by its ammo-acid sequence, changes in this sequence can cause structural changes which can provide novel surfaces, complementary to proteins, other macromolecules or small molecules. In the case that such surfaces are complementary to the transition states of chemical reactions, the free energy of the transition state is lowered by binding of the substrate to such a surface and the reaction is catalyzed by the surface.
  • the art has employed in vivo systems, such as peptide libraries cloned as continuous epitopes into Rop [Vispo NS, 1993] or thioredoxm [McCoy J, 1992] , peptides displayed on the surface of replication competent phages such as filamentous phages [Hoess RH, 1993] or bacteria [Little M, 1993] .
  • PH domains are proteins of a sequence length of approximately 110 amino acids. They were first described as an internal sequence homology at the N-terminus and C- terminus of Pleckstrin [Haslam RJ, 1993; Mayer BJ, 1993] and subsequently more than ninety such domains were identified by sensitive sequence comparison searches, predominantly as components of proteins of eukaryotic cellular signaling pathways [Gibson TJ, 1994] , and aligned. As is usually the case in protein families of low sequence similarity, sequence insertions and deletions relative to the core alignment can be found in all loops that connect elements of secondary structure.
  • the overall structure of the PH domain is that of a seven stranded, antiparallel , bent beta-sheet, covered on one end by a C- termmal helix [Figure 1] .
  • the physiological function of PH domains is not precisely known - and may indeed vary from protein to protein - many PH domains appear to bind mositol bis- and trisphosphates with high affinity. Such binding may either effect the localization of the PH domain to phospholipid membrane surfaces, cause an allosteric effect due to second messenger binding, or both. It has been suggested that a surface epitope of PH domains, bordered by the loops AB, CD and FG, or an epitope bordered by the loops CD and EF may be responsible for such binding.
  • a surface epitope of PH domains bordered by the loops AB, CD and FG, or an epitope bordered by the loops CD and EF may be responsible for such binding.
  • the N-terminal PH domain of Pleckstrin has been mutated in its framework region to define residues important for the binding of inositolphosphates . Mutations of residues adjacent to loop AB resulted in a reduction m binding affinity towards phosphatidylmositol 4,5- bisphosphate [Harlan JE, 1995] .
  • the PH domain of beta- adrenergic receptor kinase was mutated in the helix C- termmal to loop FG to investigate binding to G-beta-gamma subunits and mositol phosphates.
  • a hemagglutmm epitope has been used as a C-termmal extension of the PH domain of the Ras exchange factor Son-of-sevenless to enable the antibody affinity preparation of the recombinant protein [McCollam L, 1995] .
  • the PH domains themselves remained unmodified.
  • a His-tag epitope has been has been used as a C- termmal extension of the PH domains of Dbl, Sos 1, IRS-1 and beta-ARK to enable the immobilized-metal-affinity chromatographic purification the recombinant protein [Mahadevan D, 1995] .
  • the PH domains themselves remained unmodified. Screening for a PH domain ligand.
  • a particularly useful and versatile system for the in vivo screening of protein-protein interactions employs two fusion proteins respectively comprising DNA binding and transcription activating domains as exemplified in the interaction trap [Fields S, 1989] and two hybrid [Estojak J, 1995] systems. It has been reported that natural PH domains have been used in an interaction trap system to identify possible proteinaceous ligands [Ferguson KM, 1994] . Without specifying details, the authors state that no ligands were obtained and no binding to a proteinaceous ligand could be demonstrated and that this could be interpreted as that the PH domain would not bind to proteins or was not functional in this screening system.
  • the present invention provides a structural framework for the display of continuous and discontinuous surface epitopes in a context of antiparallel beta strand secondary structure which does not require the oxidation of disulfide bridges for functional expression and which folds independently, is highly stable, can be efficiently expressed recombinantly and efficiently refolded.
  • modified sequences of PH domains may include restriction sites suitable for the efficient cloning of random oligonucleotide libraries into the structural context of the domain through methods of directional cloning or PCR.
  • sequence changes may code for amino acid changes that facilitate the handling of the domain, for example by improving the domain stability, improving the folding properties of the domain, improving its resistance to oxidation or aggregation, improving its protease resistance, facilitating or improving the purification of the protein, altering its properties of immunogenicity, or modifying the proteins capacity for being localized m specific cellular compartments.
  • the above functions may be achieved through the addition of N- terminal or C-terminal extensions to the protein sequence.
  • three surface loops may be modified singly, or in combination to provide novel continuous or discontinuous surface epitopes .
  • sequences so obtained may be used as lead structures for small molecular weight compounds and we describe an approach to the preparation of small molecular weight ligands derived from such sequences, which are conformationally constrained in a way comparable to their conformational constraint in the original context of the protein structure.
  • the present invention relates to a nucleic acid vector molecule, characterized in that it comprises a first non- natural nucleic acid sequence encoding a polypeptide, wherein the topology of the tertiary fold of said polypeptide is homologous to that of a Plcckstrm-Homology (PH) domain, characterized in that said non-natural nucleic acid sequence, as compared to corresponding natural nucleic acid sequences,
  • PH Plcckstrm-Homology
  • (a) comprises, in at least one of the nucleotide sequences encoding a loop of the PH domain, at least one altered nucleotide, wherein said at least one altered nucleotide confers an alteration m the corresponding amino acid and said alteration (s) m said corresponding amino acid(s) confer (s) a gain of function;
  • (b) comprises, in at least one of the nucleotide sequences encoding a loop of the PH-domain or the regions directly adjacent thereto a sequence representing one or more endonuclease restriction sites; or/and (c) confers to the encoded polypeptide an improved stability, improved folding properties, an improved resistance to oxidation, aggregation or an improved protease resistance, facilitated or improved purification, altered properties of immunogenicity, and/or the capacity for being localized in specific cellular compartments.
  • the vector molecule of the present invention is characterized in that said loop is loop AB, CD or FG.
  • the vector molecule of the present invention is characterized m that said PH domain is the cytohesin 1 or cytohesin 2 domain.
  • the vector molecule of the present invention is characterized m that it additionally comprises, adjacent to the 5'- or 3 ' -end of said first non-natural nucleic acid sequence, at least one further nucleic acid sequence encoding an extension of the N- or C-terminus of the corresponding polypeptide that facilitates or improves the purification or detection of said polypeptide, or aids in targeting said polypeptide to a specific location.
  • the invention further relates to a recombinant nucleic acid vector molecule comprising
  • the (recombinant) vector molecule of the present invention is characterized in that said nucleic acid is DNA.
  • the recombinant vector molecule of the invention is characterized in that said one or more ammo acid sequences encoded by said at least one second nucleic acid sequence replace at least a part of at least one loop, preferably loop AB, CD and/or FG.
  • the recombinant vector molecule of the invention is characterized in that said one or more ammo acid sequences encoded by said at least one second nucleic acid sequence are inserted into at least one loop, preferably loop AB, CD and/or FG.
  • the recombinant vector molecule of the invention is characterized in that said one or more ammo acid sequences encoded by said at least one second nucleic acid sequence form or contribute to the forming of a continuous epitope.
  • the recombinant vector molecule of the invention is characterized m that said one or more amino acid sequences encoded by said at least one second nucleic acid sequence form or contribute to the forming of a discontinuous epitope.
  • the recombinant vector molecule of the invention is characterized in that said one or more ammo acid sequences encoded by said at least one second nucleic acid sequence comprise ammo acid sequences that represent or contribute to the representation of a lead structure, a (poly) peptide with ligand binding capabilities, a (poly) peptide with substrate-modifying capabilities, or a (poly) peptide capable of interacting with a catalyst or reactant .
  • the present invention also relates to a library of recombinant vector molecules comprising a plurality of recombinant vector molecules according to the present invention.
  • the present invention furthermore relates to a host transformed with the recombinant vector molecule according to the present invention.
  • the host of the present invention is characterized in that it is E. coll, a yeast cell, preferably S. cerevisiae, an insect cell or a mammalian cell. Further the present invention relates to a plurality of hosts according to the present invention, transformed with a library of the present invention.
  • the present invention furthermore relates to a (poly)peptide encoded by the vector molecule according to the present invention or produced by the host according to the present invention or the plurality of hosts of the present invention.
  • Said polypeptides may also be chemically synthesized and have the same amino acid sequence as the (poly)peptide encoded by the vector molecule of the invention.
  • Said (poly)peptide may further have an amino acid sequence that comprises compounds that do not naturally occur m (poly)peptide chains, preferably stereoisomers, D-amino acids, protemous ammo acids or ⁇ amino acids but being otherwise identical to the above- recited polypeptides.
  • the present invention relates to a library comprising said (poly) peptides .
  • said library comprises said chemically synthesized (poly) peptides, which may or may not have non-naturally occurring compounds contained therein.
  • the chemical synthesis of the (poly) peptides may, for example, be effected by utilizing fragment condensation techniques or one step procedures which result in whole (poly)peptide chains.
  • the non-natural peptide compounds may be molecules that are susceptible to techniques of peptide synthesis.
  • the libraries referred to above may be effected by methods of combinatorial chemistry.
  • the construction may be effected by split synthesis or by utilizing building blocks consisting of fixed peptide sequences and sequences -
  • a small molecular weight ligand is derived from the (poly) peptide according to the present invention.
  • the small molecular weight ligand according to the present invention is characterized in that it is conformationally constrained and is preferably a cyclopeptide .
  • the present invention additionally relates to a method for the generation of a library according to the present invention, characterized m that it comprises the insertion of said second nucleic acid sequences encoding a plurality of ammo acid sequences into said first or corresponding natural nucleic acid sequences.
  • the invention also relates to a method of screening a library according to the present invention comprising
  • the present invention also relates to a method of evolving or screening the library according to the present invention comprising
  • said method of the invention is characterized in that it employs steps that physically couple the genetic information of said second nucleic acid sequences with the expressed and screened (poly)peptides .
  • step (a) is characterized in that said expression in step (a) is effected via phage display techniques.
  • the expressed polypeptides or the coupling of genetic information of the second nucleic acid sequences and the expressed polypeptides may be preferable to display the expressed polypeptides or the coupling of genetic information of the second nucleic acid sequences and the expressed polypeptides on the surface of host cells, for example, utilizing outer membrance proteins, subunits of surface appendages or secreted proteins.
  • the method according to the invention is characterized in that said screening is effected by via techniques that mediate the coupling of genetic information and expressed polypeptide by DNA- bmding domains fused to said polypeptides encoded by said second nucleic acid sequences, wherem said DNA-bmdmg domains bind to said vector molecules according to the invention, or wherein said screening is effected by polysome display techniques or by interaction trap techniques .
  • DNA-bindmg domain is the tet repressor molecule or the lac repressor molecule.
  • the present invention further relates to a kit comprising the vector molecule according to the present invention and/or a nucleic acid vector molecule, characterized in that it comprises a first non-natural nucleic acid sequence encoding a polypeptide, wherem the topology of the tertiary fold of said polypeptide is homologous to that of a Pleckstrm-Homology (PH) domain, characterized in that said non-natural nucleic acid sequence, as compared to corresponding natural nucleic acid sequences,
  • PH Pleckstrm-Homology
  • (a) comprises, in at least one of the nucleotide sequences encoding a loop of the PH domain, at least one altered nucleotide
  • (b) comprises, in at least one of the nucleotide sequences encoding a loop of the PH-domain or the regions directly adjacent thereto a sequence representing one or more endonuclease restriction sites; or/and (c) confers to the encoded polypeptide an improved stability, improved folding properties, an improved resistance to oxidation, aggregation or an improved protease resistance, facilitated or improved purification, altered properties of immunogenicity, and/or the capacity for being localized in specific cellular compartments.
  • the present invention also relates to a pharmaceutical composition or vaccine comprising the (poly) peptide according to the present invention or a ligand derived therefrom.
  • the present invention also relates to the use of the vector molecule according to the present invention or the recombinant vector molecule according to the present invention for gene therapy.
  • a library is a plurality of sequences. Generally this plurality of sequences will be part of a sequence comprising common parts, represented by all or almost all molecules of the library, and unique parts, represented only by a single molecule of the library or represented only rarely.
  • Loop As used herein, a loop is the sequence of ammo acids bridging two elements of secondary structure. The loops of Pleckstrin Homology domains are defined m Figures 1 and 2.
  • Framework As used herein, the framework of a protein consists of those residues that are not part of the loop sequence .
  • the term functional is used to indicate a function of a proteir, acquired or significantly enhanced as a result of the methods of the invention. Typically this function will be an (improved) binding of a ligand.
  • Function of a protein or protein domain as used herein indicates a detectable effect of the interaction of the protein or protein domain with another molecule. Typically, function is intended to mean the binding of a ligand.
  • Gam of function as used herein is an acquisition of a novel interaction or an increased effect of an existing interaction of the protein or protein domain m contrast to a loss of function such as the loss of a phosphorylation site.
  • a domain is a structurally defined subunit, usually an autonomously folding polypeptide. Frequently, domains are observed as functional modules in larger proteins .
  • restriction site is a nucleotide sequence which will be recognized as a cleavage site by a restriction endonuclease .
  • a fusion protein is a hybrid protein, constructed to contain sequences from at least two different proteins or peptides.
  • a surface epitope is an ammo acid or a set of amino acids, which is a subset of a protein, which contribute to a contiguous part of the solvent accessible surface of that protein.
  • a continuous epitope is a surface epitope formed by amino acids placed sequentially m the primary structure of a protein.
  • discontinuous epitope is a surface epitope formed by two or more (nonsequential) continuous epitopes
  • a reporter gene is a gene which will confer upon a host a detectable phenotype, if a certain condition whithm said host is present.
  • a reporter gene can oe actively expressed as a result of a function posessed by a representative of an epitope library, such as tight binding to a ligand.. Displayed.
  • a molecule or part of a molecule is displayed if it contributes to the accessible surface of a molecule in such a way that interactions with other molecules are possible.
  • a phenotype is any detctable change in appearance, function or behaviour of a host cell.
  • a phenotype can be the color of a colony, the growth characteristic of a cell, the capacity of a cell to catalyze a chemical reaction or to bind compounds that can be detected by physical or other means, with high affinity and thus concentrate them in the cell or its immediate surrounding. Examples of these and other detectable phenotypes are well known to one of skill in the art.
  • the methods and compositions of the present invention permit the utilization of a novel structural principle for the display of continuous and discontinuous surface epitopes in a protein.
  • This protein structural framework does not require the oxidation of disulfide bridges for functional expression.
  • the novel surface epitopes are displayed in a context of antiparallel beta strand secondary structure particularly suitable for the construction of small molecular compounds based on functional sequences of modified PH domains.
  • nucleic acid vector molecule which is modified to allow the generation of such proteins with genetic engineering methods.
  • modifications may consist of nucleotide sequence changes which facilitate the generation of novel proteins in the structural framework provided by this invention, while retaining the desirable characteristics of the protein template.
  • modifications may introduce new restriction sites or delete existing restriction sites in the nucleotide sequence of the protein, with the aim of providing unique restriction sites adjacent to the regions of the sequence which are to contribute to novel surface epitopes.
  • an oligonucleotide primer with a stretch of randomized nucleotides, and restriction sites near the 3' and 5' end, is converted into a double stranded oligonucleotide after annealing with a complementary primer.
  • This oligonucleotide is then cleaved with the appropriate restriction endonucleases and ligated into the prepared vector molecule, where the randomized nucleotides encode a plurality of ammo acid sequences inserted m frame into the structural template molecule.
  • Modifications to the nucleotide sequence of the structural framework may change the protein amino acid sequence and introduce properties, desirable for technical and other reasons . It may be advantageous to increase the stability of the modified PH domain framework beyond that which is found in naturally occurring domains. Some of the peptide sequences intended to form new surface epitopes in the structural context of the modified PH domain framework may prove to be destabilizing and this may be compensated by increased levels of stability of the framework. Additionally, some uses of novel domains may require stability towards denaturant agents and/or high temperatures. Again, such uses may be facilitated by the increase of folding stability.
  • Such engineering to improve the thermodynamic stability of the modified PH domain framework may at the same time improve the folding properties of the domain.
  • Such improved folding properties may also be achieved through other mutations and may be desirable for technological reasons.
  • the removal of slow folding steps which may lead to the accumulation of aggregation-prone folding intermediates, may reduce the propensity for the domain to aggregate during refolding.
  • Slow folding steps have been reported for proteins that require the isomerization of a peptide-prolme bond from the energetically favored trans-conformation into a cis- conformation which may be necessary for structural reasons.
  • An improved resistance to oxidation of disulfide linkages within or between modified PH domains may be desirable for technical or other reasons and may be achieved through the partial or complete replacement of cysteine residues with suitable other ammo acids such as serme, alanme or valine.
  • modified PH domains towards aggregation and an increased solubility may be desirable for technical or other reasons. For example, this may improve the shelf life of compositions comprising modified PH domains, improve production processes including modified PH domains and the production of the domains themselves and improve the usefulness of modified PH domains in pharmaceutical compositions. This can be achieved through changes to the modified PH domain framework sequence, for example through the substitution of hydrophobic residues by other ammo acids - a procedure that has been successfully applied to a respiratory syncitial virus major glycoprotem fragment [Murby M, 1995] .
  • an improved resistance towards aggregation during refolding or afterwards and an increased solubility of modified PH domains may be achieved through provision of glycosylation sites, phosphorylation sites and/or charged residues, through mutagenesis of appropriate surface residues.
  • An improved resistance towards proteolytic cleavage and degradation of modified PH domains may be desirable for technical reasons and increase the usefulness of the domains in vivo and in vitro.
  • specific recognition sites are known and may be changed in the sequence of modified PH domains to reduce their sensitivity towards such proteases .
  • An increased susceptibility towards limited proteolytic cleavage by specific proteases, or towards chemical cleavage may be desirable, to obtain peptides from the expressed domains.
  • protease recognition sites such as that of activated Factor X, Ile-Glu-Gly-Arg, or Thrombm, Leu-Val-Pro-Arg-Gly-Ser, or the ammo acid methionine may be engineered to occur m the ammo acid sequence on both sides adjacent to a loop region.
  • peptides comprising the sequence between these residues may be obtained by cleavage of the modifies PH domains with the appropriate protease, or by treatment with cyanogen bromide under conditions well know to one of skill m the art, whichever may be appropriate for the chosen modification.
  • affinity chromatographic techniques for a facilitated purification of modified PH domains may be desirable for technical reasons For example, this may be achieved through the introduction of a surface epitope comprising histidine residues to coordinate bivalent metal cations which can subsequently be complexed by a suitable affinity matrix. Such a procedure has been successfully applied to retmol binding protein [Muller HN, 1994] .
  • modified PH domains intended for in vivo use or comprising pharmaceutical compositions may be reduced through the substitution of sequence epitopes foreign to the species in which the modified PH domains are to be used, with such sequence epitopes native to the species, or with residues which may be less lmmunogenic for other reasons.
  • de novo design of sequences, or the construction of hybrid PH domains comprising sequences of at least one other naturally occurring protein or proteins may be employed.
  • Such a procedure has been successfully applied to antibody variable domains [Roguska MA, 1994] .
  • proteins are transported into specific cellular compartments and the use of modified PH domains may require such specific transport. This may be effected by the introduction of appropriate sequence signals. Examples include nuclear localization signals such as the adenoviral sequence Lys-Arg-Pro-Arg-Pro, or retention signals for the endoplasmic reticulum such as the C-terminal sequence Lys- Asp-Glu-Leu.
  • N- or C- terminal fusion with sequences comprising residues which provide stabilizing interactions to the framework.
  • Such fusion of N- or C-termmal sequences to the modified PH domain framework sequence may at the same time, or independently, improve the folding properties of the domain. For example an improved resistance towards aggregation during refolding or afterwards and an increased solubility of modified PH domains may be achieved through provision of N- or C- terminally fused sequence extensions comprising charged residues, phosphorylation sites and/or glycosylation sites.
  • N- or C- terminal fusion with protease inhibitory sequences or protease inhibitors
  • Affinity chromatographic techniques for a facilitated purification of modified PH domains may be employed after fusion with N- or C- terminal sequences comprising histidine residues [Lindner P, 1992] , epitopes, recognized by specific antibodies [Evan GI , 1985] , epitopes binding streptavidme [Schmidt TGM, 1994] , the ribonuclease S-peptide [Kim JS, 1993] , stretches of charged residues, fusion proteins such as for example Glutathione S-transferase [Smith DB, 1988] , or the use of other techniques kown to one of skill m the art.
  • modified PH domains may be effected by N- or C- terminal fusion with appropriate sequence signals such as the adenoviral nuclear localization sequence Lys-Arg-Pro-Arg- Pro, or the C-termmal endoplasmic retention signal sequence Lys-Asp-Glu-Leu.
  • Other applications of modified PH domains may require binding the domains to specific targets. This can be achieved through N- or C- terminal fusions with peptides or proteins specifically binding such targets such as antibodies or DNA binding domains.
  • modified PH domains may require binding functional peptides or proteins, such as enzymes or inhibitors, to specific targets via the affinity provided by the modified PH domain itself and this can be achieved through N- or C- terminal fusions of the functional peptides or proteins to an appropriately constructed or selected modified PH domain.
  • functional peptides or proteins such as enzymes or inhibitors
  • epitope modifications may be performed in surface loops AB, CD and FG of a suitable modified PH domain, singly or m any combination, such epitope modifications retaining the desirable properties of the original domain such as folding independently, being highly stable, being suitable for efficient recombinant expression and efficiently refolding.
  • Such epitope modifications may provide conformationally constrained continuous sequence epitopes. Continuous sequence epitopes can provide ligands with dissociation constants of micromolar magnitude or less, and their appropriate conformational constraint may improve the dissociation constant by a large amount .
  • conformational constraint is provided by the stable anchoring of surface loops AB, CD, EF and FG m antiparallel beta-strands of the modified PH domain framework.
  • discontinuous epitopes Much greater binding affinity than that of continuous sequence epitopes may be provided by discontinuous epitopes. This may significantly increase the number of interactions between the surface epitopes of the modified PH domain and its ligand.
  • Discontinuous epitopes may provide dissociation constants on the order of nanomolar magnitude or less and the possibility of constructing or otherwise obtaining large discontinuous surface epitopes with high affinity to a ligand is an important aspect of the invention.
  • Such discontinuous epitopes may comprise more than one of the surface loops AB, CD, EF and FG of the modified PH domain in any combination, preferably all three of the surface loops AB, CD, and FG.
  • a library or libraries of continuous sequence epitopes in modified PH domains may be constructed m the following manner. Utilizing techniques of site-directed mutagenesis known to one of skill in the art, the natural or modified nucleotide sequence for the PH domain may be altered in such a way as to introduce suitable, preferably singular, restriction sites flanking both sides of the sequence coding for surface loops AB, CD or FG. Alternatively, restriction sites in the vector or coding sequence may be deleted using said methods of mutagenesis, to make a restriction site occurring more than once in the natural sequence singular. Alternatively, a single restriction site directly adjacent to, or within said surface loop sequences may be introduced or made singular.
  • a suitable endonuclease restriction site or sites into the nucleotide sequence encoding the modified domain, that sequence may be cleaved to generate compatible ends for subsequent ligation of a randomized oligonucleotide.
  • the sequence of the randomized oligonucleotide itself may be introduced through site-directed mutagenesis, preferrably, but not necessarily, at the same time deleting an existing natural or previously introduced non-natural, singular restriction site to allow restriction selection of the mutant genotype.
  • Said oligonucleotide is designed to comprise the same or compatible restriction endonuclease sites and to additionally comprise a stretch of nucleotides that have been synthesized to provide degenerate sequences, i.e.
  • a degenerate codon of the sequence NNB will specify 48 different codons, all not ending in A.
  • the use of such randomized codons will reduce the probability of introducing a stop codon from 0.047 (or one in 21 codons) to 0.021 (or one in 48 codons) , thus the number of random positions that may be generated while not letting the probability of introducing a stop codon rise above 0.5 is increased from ⁇ 14 to - 33.
  • a second nucleotide, complementary m sequence to the 5' end of said randomized oligonucleotide is annealed to said oligonucleotide and the complementary strand is generated through the action of a suitable polymerase.
  • the efficient ligation of the randomized oligonucleotide sequences into the domain vector sequence is then effected through techniques well known to one of skill in the art, ultimately producing a library of sequences of modified PH domains.
  • the library may be constructed by gene synthesis of the entire domain or parts thereof wherem the randomized segments are complemented by action of a suitable polymerase.
  • a suitable host can then be transformed with such libraries of sequences, or the library of sequences can be used directly or after amplification by the polymerase chain reaction, for further manipulation of the nucleotide sequence.
  • Libraries of sequences of discontinuous epitopes may be generated according to the above procedure by simultaneously ligatmg more than one randomized oligonucleotide sequence into a vector sequence suitably prepared m more than one sites, m a multi-fragment ligation.
  • the generation of libraries of discontinuous epitope sequences will be performed via the combination of libraries of randomized continuous epitopes using cloning techniques well known to one of skill in the art.
  • the said chemically synthesized polpeptide chain has to fold to its natural occuring three dimensional structure.
  • a favourable feature for a polypeptide chain to fold spontanously under physiological conditions is the lack of disulfide linkages because the incorrect connection of free cysteine residues often results m non-folded, aggregating proteins.
  • the naturally occuring PH domains do not posess any disulfide linkages because their rigid structure of antiparallel ⁇ -strands does not need a stabilization by disulfide bridges.
  • the special technical interest of a modified PH domain in whicn the naturally occuring free cysteine residues are replaced as described before is to be mentioned
  • the synthetic libraries provide the advantage of incorporation of non-natural amino acids into the peptide chain e g D-ammo acids, stereoisomers of natural ammo acids or any other compound which is susceptible to the coupplmg chemistry. Screening for increased affinities to a ligand.
  • a combinatorial library of surface epitopes, constructed with an appropriately large plurality of sequences of modified PH domains will contain among these sequences those, that will posses an increased affinity towards a desired ligand.
  • methods are described that will allow to discriminate between sequences binding a certain ligand and those that do not .
  • such methods and procedures rely on the physical coupling of the expressed protein with its nucleic acid vector molecule, for example through the non-covalent binding of the expressed protein sequence with its vector, the identification of functional sequences within a suitable host cell, that carries the vector sequence or through the display of the expressed protein on the surface of the suitable host-cell or virus carrying the encoding nucleic acid sequence.
  • the library is prepared using a vector molecule that comprises a modified PH domain sequence, which is fused to the N- or C- terminus of a peptide or protein that can bind a specific DNA sequence with high affinity and a slow kinetic off-rate such as for example the lac-repressor
  • this procedure as applied to screening libraries of sequences of modified PH domains, may entail creating a library of vector molecules in such a way, that the nucleic acid sequences encoding the library of modified PH domains is fused to the 5 1 end of a second nucleic acid sequence encoding a transcriptional activator domain, for example ammo-acids 768-881 of the yeast GAL4 protein.
  • a suitable host is modified genetically to contain withm its chromosomal DNA, or on a separate plasmid, a constituitively expressed fusion protein consisting of a DNA binding domain, such as amino acids 1- 147 of yeast GAL4 , or the DNA binding domain of the bacterial protein LexA, fused to a sequence encoding the target protein for which it is desired to find modified PH domain sequences from the library, that bind to said proteins.
  • a constituitively expressed fusion protein consisting of a DNA binding domain, such as amino acids 1- 147 of yeast GAL4 , or the DNA binding domain of the bacterial protein LexA, fused to a sequence encoding the target protein for which it is desired to find modified PH domain sequences from the library, that bind to said proteins.
  • a reporter gene On a chromosome of the host cell, or on a second vector molecule, a reporter gene is located which can be bound with high affinity by the DNA binding domain of the fusion protein mentioned above, and which is followed by a sequence encoding an enzyme, other protein, peptide or sequence functional when transcribed into RNA, the expression of which confers upon the host cell a certain phenotype.
  • Such phenotype may be a property of the host cell which may become evident upon inspection, such as for example a color change effected through the enzymatic action of beta-galactosidase, or may confer upon the host cell a growth advantage under certain conditions of limiting nutrients such as the LEU or Ura locus of yeast cells, or confer upon the cell resistance against compounds normally toxic to the cell, such as the enzyme ammoglycoside-phosphotransferase conferring resistance against the gentamycm derivative antibiotic G418.
  • the modified PH domain carrying the functional surface epitope will non-covalently attach to the desired ligand molecule, which itself is bound to the DNA sequence upstream of the reporter gene via the fused DNA binding domain; thus the transcriptional activator domain which is fused to the modified PH domain will activate the transcription of the reporter gene and the detectable phenotype is expressed.
  • the actual screening step can be preceded with a purification step, in which a host is used that has been genetically prepared m exactly the same way as the host used in the screening, but lacking the actual protem-ligand against which affinity is to be screened, and replacing the detectable phenotype with one which inhibits growth.
  • a purification step in which a host is used that has been genetically prepared m exactly the same way as the host used in the screening, but lacking the actual protem-ligand against which affinity is to be screened, and replacing the detectable phenotype with one which inhibits growth.
  • elements from the library which provide the transcriptional activation via non-specific interactions, or those that interfere with cellular functions and thus by themselves inhibit growth, are depleted from the library.
  • this purified library may be used as described above.
  • a third example of screening modified PH domains employs phage display technology.
  • the aim of the method is to provide fusion proteins comprising an N-termmal modified PH domain fused to the gene III or gene VIII product of M13, FI or similar filamentous, single-stranded DNA phages.
  • This fusion protein is either encoded in the phage-genome, or in a phagemid vector.
  • phages contain many copies of the desired protein, in the latter case, after infection of the host cell with helper- phage, few, or single copies of the fusion protein can be found displayed on phage hull particles that have packaged the phagemid single stranded vector.
  • the genetic information encoding a modified PH domain is contained in the phage particles and the fusion protein is displayed on the surface of said phages.
  • a library of vector sequences with randomized PH domain surface epitopes is constructed and the PH domain is cleaved from said vectors with the appropriate restriction endonucleases.
  • a phage genome or phagemid vector is prepared by mutagenesis to contain compatible restriction sites, in frame at the 3' end of the DNA sequence encoding the gene III protein.
  • Double stranded RF-DNA is prepared from the phage, or the phagemid propagated, and the DNA is cleaved with the appropriate restriction endonucleases to provide compatible ends for the subsequent ligation of the library of modified PH domains sequence fragments.
  • the resulting RF-DNA vectors or phagemids are transformed into suitable E.
  • the single-stranded DNA molecule encoding the modified PH domain will be encapsulated with an assembly of products of gene III and gene VIII and fusion proteins comprising the modified PH domain itself in the resultant phage particle.
  • the supernatant containing the phage particles can be directly used or the particles concentrated vie Ultraflltration or precipitation. These particles are incubated with the desired ligand molecule, preferably covalently linked to a column matrix.
  • a plurality of sequences comprising modifications in more than one epitope is cleaved with appropriate restriction endonucleases and the resultant fragments, each individually expected to contribute towards binding the ligand in some way, are mixed and stochastically religated to provide new and potentially even more functional epitopes from combinations of sequence epitopes.
  • a plurality of sequences of modified PH domains can be amplified with the polymerase chain reaction under conditions that will introduce a high percentage of errors into the amplified sequence, thus effecting near-random point mutations over a desired sequence range, which can either extend over a single epitope, a combination of epitopes or over the whole domain.
  • individual loop regions are excised from the sequence of the pluralities of modified PH domains with demonstrated ligand capabilities, which are subsequently replaced with libraries of oligonucleotides comprising randomized sequence.
  • the present invention is also useful for the production of large amounts of proteins comprising continuous or discontinuous epitopes previously identified as possessing a specific function in a screening assay.
  • the nucleic acid sequence encoding the domain is cloned downstream of a strong promoter in a suitable bacterial expression vector, an example being the T7 promoter in the vector pPHCYlLl, and the expressed material is recovered from the host after induction using standard techniques well kown to one of skill in the art.
  • the protein can either be used to perform a certain function by itself, or a continuous peptide epitope can be cleaved from the protein, utilizing specific protease cleavage sites as specified above. After cleavage, the peptide may be purified from the proteases and the rest of the structural template utilizing techniques well known to those of skill in the art, for example HPLC.
  • Screened or evolved, functional, modified PH domains may be utilized in the design of lead structures for the generation of small molecule ligands.
  • single continuous epitopes may be synthesized chemically or prepared as described above and subjected to targeted or combinatorial chemical modifications with the aim of providing an even further improved function/increased functionality, or desired properties, such as for example increased permeability into the cellular cytoplasm, or preferred localization in specific organismic or cellular subcompartments, increased bioavailability, increased stability after peroral mgestion of the compound, increased or decreased clearance from body fluids, combination with toxins, which result in a targeted delivery of such toxins, combination with radiochemicals or spin labels to allow targeting of these markers in the course of diagnostic procedures.
  • compositions and gene therapy are provided.
  • compositions described above may be parts of and/or active principles in pharmaceutical compositions. They may also be parts of and/or active principles in the preparation of vaccines.
  • modified PH domains may be encoded in an expressible form on a suitable vector for gene therapy, and may be expressed m targeted cells whereupon a functional modified PH domain may become active.
  • Figure 1 illustrates the folding topology which defines a PH domain. Secondary structural elements are shown as arrows (beta strand) or helix (alpha helix) and loop AB connecting beta-strands A and B, loop CD connecting beta- strands C and D and loop FG connecting beta-strands F and G are labeled. The N and C terminus of the domain are also labeled.
  • Figure 2 illustrates a sequence alignment of the structurally characterized PH domains of rat dynamin (Dynl_Rat) and human spectrm (Spcnmr_Hum) , as well as the PH domains of human beta-adrenergic receptor kmase (Arkl_Human) , human Vav protein (Ph-Hvav) , human cytohesin 1 (Cythl_Huma) , human cytohesin 2 (Cyth2_Huma) and human Ras protein (Ph-Rasa-Hu) .
  • Figure 3 shows the position and approximate relative size of the genetic elements on plasmid pPHCYl .
  • Figure 4 illustrates the DNA sequence (SEQ ID NO: 11) of the expression plasmid pPHCYlLl for the expression of the modified PH domain sequence PHCY1L1 described in Example 1, and the ammo acid sequence (SEQ ID NO: 12) of the modified PH domain sequence PHCY1L1 contained therein.
  • Figure 5 illustrates the DNA sequence (SEQ ID NO: 13) of the random oligonucleotide primer used for the generation of the library of peptides inserted into the PHCY1L1 domain described in Example 1 .
  • Figure 6 illustrates the DNA sequences (SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO: 5, SEQ ID NO:7 and SEQ ID NO: 9) of the m- frame mutants with which stability measurements were performed as described in Example 1.
  • the sequences listed here begin with the nucleotide corresponding to nucleotide 139 of the plasmid sequence (SEQ ID NO: 11) , codon 12 of the PH domain sequence.
  • Figure 7 summarizes the stability measurements for progenitor and mutant PH domains.
  • Delta G is the free energy of folding of the domains m PBS at 25 degree C given m kJ/mol .
  • Figure 8 illustrates the DNA sequences of the oligonucleotides for the replacement of cysteine residues as described in Example 4.
  • the oligonucleotides complement the coding strand of the expressed domain and are thus designated "reverse".
  • the mutations were C32A (SeQ ID
  • Figure 9 shows the position and approximate relative size of the genetic elements on plasmid pTLP2.
  • Figure 10 illustrates the relative fluorescence of Green Fluorescence Protein in a suspension of bacterial cells, plotted against the concentration of anhydrotetracyclme in the culture medium. The titration of both the wildtype tet- repressor (filled circles) and the tet-repressor-PH-domain fusion protein with anhydrotetracyclme results in equal levels of induction of fluorescence. This demonstrates that the wildtype repressor and the fusion protein have the same affinities for binding the inducer as well as for binding the operator site.
  • Figure 11 shows the sequence of the fixed reverse primer, complementary to the coding strand, used in the randomization experiment described in Example 6. It also shows the sequence and target base compositions for the randomized oligonucleotides used in the same example. Looplrm-ls is desigend to randomize Loop AB and Loop2rm-s is designed to randomize Loop CD.
  • Figure 12 shows the comparison of loop sequences from the randorized library described in example 6.
  • the position of the nucleotides refer to Seq ID 21, the position of the corresponding amino acids refer to Seq ID 22.
  • the mutated nucleotides are underlined, the corresponding ammo acids are in italics.
  • Figure 13 illustrates the nucleotide and amino acid sequence of the CR6 peptides inserted in three loop sequences of tne PHCY1 synthetic PH domain. Restriction enzyme sites are shown in cold. Underlined sequences represent the oligonucleotides used for the PCR reactions. Underlined ammo acid sequences represent the CR ⁇ peptides.
  • Figure 14 shows the expression pattern of synthetic PH doma ⁇ n/B42 transactivator domain fusion proteins in yeast cells.
  • Figure 15 shows a comparison of beta-galactosidase activities from wt and mutant synthetic PH domains, m which the CR6 peptide was inserted into loop sequences. Beta-galactosidase activities were determined from four independently isolated clones from each construct. Given are the men values which showed a standard deviation between 5 to 15%.
  • Example 1 High level expression and folding stability measurement of a synthetic PH Domain
  • a cDNA clone of the human cytohesin 1 protein (formerly known as sec7 homology protein, Genbank ID: Humsec7hom) , obtained from human natural-killer T lymphocytes was obtained by standard procedures.
  • the PH domain from this cDNA was amplified in a PCR reaction, utilizing primers to generate a synthetic PH domain comprising a start codon and non-natural N-terminal ammo acid for the generation of an Nco I restriction site, a non-natural glycme and hexahistidine tag on the C-termmus forming a BstE II restriction site, a stop codon and an EcoR I restriction site.
  • the resultant synthetic PH domain was cloned into the expression vector pRSet5d (GenBank X54205) under control of the T7 promoter to obtain plasmid pPHCYl ( Figure 3, 4) (SEQ ID NO: 13) .
  • a protocol for preparative scale expression and purification was used in the following way: 2 L of culture medium was inoculated with an overnight culture of E.coli BL21 (DE3) , transformed with the appropriate mutant plasmids. The cells were induced through addition of ImM IPTG at an OD(600 nm) of approximately 0.6 and grown for a further 3 h, when the cells were pelleted by centrifugation. Cell pellets were washed once with PBS
  • the eluted protein was dialyzed against PBS containing 1 mM DTT and brought to 5 mM DTT, 0.02% sodium azide, before storage at 4 degrees C.
  • the yield of the isolated synthetic PH Domain was 25 mg of purified product per L of bacterial culture, equivalent to 20 % of the whole soluble cell protein.
  • Free energy values of folding were calculated assuming a linear relationship of the free energy of folding on the denaturant concentration, and applying a pre- and post-unfolding baseline correction in a non-linear least-squares fit of the observed fluorescence to the denaturant concentration, assuming a two-state model of folding.
  • the stability was found to be -39 ⁇ 1.6 kJ/mol , which is surprisingly high for an isolated protein domain. This makes the protein a good starting point for random mutagenesis experiments and for further optimizations of a suitable rigid backbone and of properties for biotechnological handling.
  • a synthetic PH domain based on the sequence of the human protein cytohesin 1 can be expressed to high levels in a bacterial host, folds stably, and can be purified in a single step from a bacterial extract via an affinity tag genetically fused to the C-termmus.
  • Example 2 A library of sequences m loop AB of a synthetic PH domain based on the cytohesin 1 sequence
  • site directed mutagenesis was used to generate a library of domain sequences using oligonucleotide CylllRMas (SEQ ID NO: 15) ( Figure 5) .
  • the oligonucleotide was designed to generate randomized sequences in loop AB, two new restriction sites 5' and 3' of the coding sequence, and to eliminate the unique restriction site of BsmB I . Phagemid single strand
  • DNA was prepared from pPHCYl in the dut " ung " strain CJ236
  • Example 4 Functional integrity of a fusion protein of tet- repressor and a synthetic PH domain
  • the synthetic PH domain of Example 3 was genetically fused to the C-Termmus of the tet-repressor class D sequence.
  • This fusion protein allows site specific binding of a protein comprising a synthetic PH domain to a DNA sequence with high affinity.
  • the sequences of the tet- repressor coded by the plasmid pASK75 [Skerra A, 1994] and of the synthetic PH domain described in example 3 were amplified in a PCR reaction.
  • the utilized primers created an overlapping linker sequence GCT CCG GCA GCT GCT AAA CAG GAA GCT GCA CCG GCT GCA coding for the peptide APAAAKQEAAPAA.
  • This linker connected the last ammo acid of the tet-repressor directly to the first ammo acid of the PH domain.
  • An NspV-site was introduced into pASK75 by a standard site directed mutagenesis procedure, 3' of the tet-repressor sequence and the amplification product was cloned into pASK75 using the Nsil site inside the tet- repressor and the NspV-site 3'of the PH domain.
  • the fusion protein is expressed from a polycistronic message, constltuitively transcribed from the beta-lactamase promoter gene.
  • the gene for the green fluorescent protein including a ribosome binding site has been Xbal/Hmdlll cloned downstream of the tet- operator/tet-promotor region replacing the ompA sequence, and the strep tag of pASK75.
  • This plasmid (Seq ID 23) was used to transform competent E. coli JM109 cells (Stratagene GmbH, Heidelberg, Germany) .
  • E. coli JM109 cells were grown to an OD(600 nm) of 0.6 and induced with different concentrations of anhydrotetracyclme. After 3 hours of expression 1 mL aliquots of cells were harvested by centrifugation, washed once with phosphate buffered saline and resuspended in 2 mL of PBS. The cell suspensions were adjusted to an OD(600nm) of 0.5 and were submitted to fluorescence analysis at 22°C using an excitation wavelength of 395 ⁇ 2.5 nm and an emission wavelength of 510+2.5 nm.
  • Figure 10 shows the comparison of the relative fluorescence of cultures of E.
  • Loops AB and CD were randomized at the same time. Oligonucleotides were synthesized on an Eppendorf Ecosyn D300 synthesizer to comprise a part of the coding PH domain sequence flanking a randomized portion in the regions coding for loop ammo acids. Each loop was separately randomized by performing a PCR reaction using an oligonucleotide coding for a randomized sequence as forward primer, in combination with a fixed reverse primer complementary to the 3 '-end of the PH-domam sequence.
  • the resulting products were restricted by the single cutting restriction endonucleases Aflll and Accl (Loop AB) respectively Accl and Xhol (Loop CD) creating compatible cohesive ends. Fragments of the expected size were gel purified and ligated, resulting in a non-interrupted PH- domam sequence corresponding to bp 3153 - 3482 of SEQ ID No. 21.
  • the ligation products were amplified by PCR using the randomized forward primer of loop AB and the fixed reverse primer.
  • the Sequences and target copmositions of the synthesized oligonucleotides are given m Figure 11.
  • the resulting PCR product (500 ng) was restricted with the enzymes Aflll and Xhol, gel purified and ligated to the compatibly restricted and purified vector (3.5 micro g) .
  • the ligation product was dialyzed and used for electroporation of competent E. coli BL21(DE3) .
  • the resulting library consists of 2*10 6 individual clones which are assumed to be independent .
  • Figure 12 shows the loops sequences of 9 arbitrary chosen clones.
  • the observed ammo acid composition at the randomized position closely matches that expected from the target nucleotide composition of the oligonucleotides.
  • Example 6 Generation of a synthetic PH domain capable of interacting with another protein in a yeast interaction trap
  • the synthetic PH domain from plasmid pPHCYCl was amplified m a PCR reaction, utilizing primers to generate a synthetic PH domain comprising a EcoR I and Spe I restriction site and a start codon, a stop codon and a Sal I restriction site [primers PHT and PHB, figure 13] .
  • Said domain was cloned into compatible sites of the yeast expression vector pJG4-5 (Ausubel et al .
  • the other utilized the 3' primer for the said domain with the stop codon and Sal I restriction site and a internal primer designed for an Nco I restriction site, for coding of the last ammo acids of the CR6 peptide and an additional tryptophan [primers LITW and LIBW, figure 13] .
  • both resulting fragments were ligated simultaneously to the EcoR l/Xho I cleaved vector pJG4-5 to obtain the plasmid pJG- PHCR6 ( Figure 13) .
  • Plasmid pJG-PHwt and pJG-PHCR6 were transformed into the yeast strain EGY48 (Ausubel et al . ) and whole cells from two independent clones of each construct, either grown under repressed conditions in the presence of glucose or grown under induced conditions in the presence of galactose, were analyzed via SDS PAGE and Western blot with anti-HA tag antibody (Boeh ⁇ nger Mannheim, Germany) . All methods were performed using standard protocols (Sambrook et al . ) .
  • the CR6 peptide without any additional ammo acids was introduced between ammo acids 16 and 17 of loop AB, between ammo acids 41 and 42 of loop CD or between ammo acids 88 and 89 of loop EF to obtain the vectors pJGPHCR ⁇ .Ll, pJGPHCR6.L2 or pJGPHCR6.L3 ( Figure 13) .
  • the 500 ammo acid open reading frame of CBF [Amakawa R, 1993; Henkel T, 1994] was cloned into the expression vector pSHl (Ausubel et al . ) where it is genetically fused to the DNA binding domain of lex-A resulting in vector pSH- CBF.
  • Vector pSH-CBF was cotransformed with the vector pJG4- 5 (negative control) or the corresponding pJG-PH vectors into the yeast strain EGY48.
  • Four independent clones of each pJG-PH-construct were grown under inducing conditions in the presence of galactose and the beta-galactosidase activity was determined according to standard procedures.
  • the PH domain can be expressed to high levels in yeast cells, as a fusion protein with the B42 transacivation domain, even when foreign peptide sequences are inserted into the original domain sequence.
  • a foreign sequence can retain properties of its progenitor protein - in our example this is the capacity to interact with the CBF protein - and that such properties are now conferred to the synthetic PH domain containing said foreign sequence.
  • the interaction of a synthetic PH domain and a target protein can be detected in a yeast interaction trap system.
  • synthetic PH domains are indeed suitable for the interaction trap based screening of peptide / protein and of protein / protein interactions. 1.
  • MOLECULE TYPE protein
  • Trp Lys Arg Arg Trp Phe lie Leu Thr Asp Asn Cys Leu Tyr Tyr
  • Lys Ala Ala lie Ser Arg Asp Gly His His His His His His His * 130 135 140
  • CTAGCGCCCG CTCCTTTCGC TTTCTTCCCT TCCTTTCTCG CCACGTTCGC CGGCTTTCCC 855
  • GTTTTTCGCC CTTTGACGTT GGAGTCCACG TTCTTTAATA GTGGACTCTT GTTCCAAACT 1035
  • Val Tyr Arg lie Ser Ala Pro Thr Pro Glu Glu Lys Glu Glu Trp lie 100 105 110
  • Lys Cys lie Lys Ala Ala lie Ser Arg Asp Gly His His His His His His 115 120 125
  • Lys Arg Arg Trp Phe lie Leu Thr Asp Asn Ala Leu Tyr Tyr Phe Glu 155 160 165
  • AAAAACTTGA TTAGGGTGAT GGTTCACGTA GTGGGCCATC GCCCTGATAG ACGGTTTTTC 975
  • CTCCCGTATC GTAGTTATCT ACACGACGGG GAGTCAGGCA ACTATGGATG AACGAAATAG 2115
  • GAA ACT CTC GAA AAT CAA TTA GCC TTT TTA TGC CAA CAA GGT TTT TCA 2842
  • AAA GCA GCC ATC TCG AGG GAC GGT CAC CAC CAT CAC CAT CAC TAG 3511 Lys Ala Ala He Ser Arg Asp Gly His His His His His His * 465 470 475
  • MOLECULE TYPE protein
  • CTTTGAGTAT ACCNNNNNNN NNNNNNNN NNNNATCATC CCTTTAGAGA ATCTGA 56
  • MOLECULE TYPE other nucleic acid
  • DESCRIPTION: /desc "synthetic oligonucleotide"
  • MOLECULE TYPE other nucleic acid
  • DESCRIPTION: /desc "synthetic oligonucleotide”
  • xi SEQUENCE DESCRIPTION: SEQ ID NO: 36:
  • MOLECULE TYPE protein

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Virology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

De manière générale, l'invention concerne des molécules protéiques ayant de nouvelles propriétés de liaison ou catalytiques. Plus spécifiquement, elle concerne la production de banques de séquences peptidiques dans le cadre d'une matrice structurelle dérivée des domaines d'homologie de Pleckstrin (PH) et l'identification des séquences possédant les propriétés désirées de liaison de ligands macromoléculaires ou constitués de petites molécules, notamment les états de transition des réactions chimiques. L'invention concerne également des petites molécules, dérivées des séquences peptidiques ainsi obtenues, possédant des propriétés de liaison de ligands comparables à celles desdits peptides dans le contexte du cadre structurel.
PCT/EP1997/002840 1996-05-31 1997-05-30 Nouvelles matrices structurelles proteiques synthetiques pour la generation, le criblage et l'evolution de surfaces moleculaires fonctionnelles WO1997045538A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP97927094A EP0912728A1 (fr) 1996-05-31 1997-05-30 Nouvelles matrices structurelles proteiques synthetiques pour la generation, le criblage et l'evolution de surfaces moleculaires fonctionnelles
JP09541632A JP2000511050A (ja) 1996-05-31 1997-05-30 機能性分子表面の生成、スクリーニング、および展開のための新規の合成タンパク質の構造の鋳型

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP96108776.4 1996-05-31
EP96108776 1996-05-31

Publications (1)

Publication Number Publication Date
WO1997045538A1 true WO1997045538A1 (fr) 1997-12-04

Family

ID=8222839

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1997/002840 WO1997045538A1 (fr) 1996-05-31 1997-05-30 Nouvelles matrices structurelles proteiques synthetiques pour la generation, le criblage et l'evolution de surfaces moleculaires fonctionnelles

Country Status (3)

Country Link
EP (1) EP0912728A1 (fr)
JP (1) JP2000511050A (fr)
WO (1) WO1997045538A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000020574A2 (fr) * 1998-10-08 2000-04-13 Rigel Pharmaceuticals, Inc. Fusion de proteines d'echafaudage avec des banques de peptides aleatoires
US6936421B2 (en) 1998-10-08 2005-08-30 Rigel Pharmaceuticals, Inc. Structurally biased random peptide libraries based on different scaffolds
US7297482B2 (en) 1998-10-08 2007-11-20 Rigel Pharmaceuticals, Inc. Structurally biased random peptide libraries based on different scaffolds
EP2382990A1 (fr) 2003-04-30 2011-11-02 Universität Zürich Procédés permettant de traiter le cancer à l'aide d'une immunotoxine
WO2014088583A1 (fr) * 2012-12-06 2014-06-12 Exxonmobil Research And Engineering Company Gènes dgat comprenant des domaines d'homologie de pleckstrine et utilisation dans des micro-organismes recombinants
US9328336B2 (en) 2012-10-16 2016-05-03 Exxonmobil Research And Engineering Company DGAT genes and methods of use for triglyceride production in recombinant microorganisms

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2102339A2 (fr) * 2007-01-12 2009-09-23 Sea Lane Biotechnologies,llc. Banques combinatoires de séquences polypeptidiques conformationnellement contraintes
AR095499A1 (es) 2013-03-14 2015-10-21 Bayer Healthcare Llc ANTICUERPOS MONOCLONALES CONTRA ANTITROMBINA b

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991005058A1 (fr) * 1989-10-05 1991-04-18 Glenn Kawasaki Synthese et isolation sans cellule de nouveaux genes et de nouveaux polypeptides
WO1991012328A1 (fr) * 1990-02-15 1991-08-22 Fowlkes Dana M Reactifs entierement synthetiques a affinite specifique
WO1992002536A1 (fr) * 1990-08-02 1992-02-20 The Regents Of The University Of Colorado Evolution polypeptidique systematique par traduction inverse
WO1993003172A1 (fr) * 1991-08-01 1993-02-18 University Research Corporation Evolution polypeptidique systematique par traduction inverse
WO1993008278A1 (fr) * 1991-10-16 1993-04-29 Affymax Technologies N.V. Banque de peptides et procede de depistage
WO1994002502A1 (fr) * 1992-07-28 1994-02-03 Genetics Institute, Inc. Fusions de peptides et de proteines pour former des molecules de thioredoxine et ressemblant a la thioredoxine

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991005058A1 (fr) * 1989-10-05 1991-04-18 Glenn Kawasaki Synthese et isolation sans cellule de nouveaux genes et de nouveaux polypeptides
WO1991012328A1 (fr) * 1990-02-15 1991-08-22 Fowlkes Dana M Reactifs entierement synthetiques a affinite specifique
WO1992002536A1 (fr) * 1990-08-02 1992-02-20 The Regents Of The University Of Colorado Evolution polypeptidique systematique par traduction inverse
WO1993003172A1 (fr) * 1991-08-01 1993-02-18 University Research Corporation Evolution polypeptidique systematique par traduction inverse
WO1993008278A1 (fr) * 1991-10-16 1993-04-29 Affymax Technologies N.V. Banque de peptides et procede de depistage
WO1994002502A1 (fr) * 1992-07-28 1994-02-03 Genetics Institute, Inc. Fusions de peptides et de proteines pour former des molecules de thioredoxine et ressemblant a la thioredoxine

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
HASLAM R J ET AL: "PLECKSTRIN DOMAIN HOMOLOGY", NATURE, vol. 363, 27 May 1993 (1993-05-27), pages 309/310, XP002030407 *
K.M. FERGUSON ET AL.: "Scratching the surface with the PH domain", NATURE STRUCTURAL BIOLOGY, vol. 2, no. 9, September 1995 (1995-09-01), WASHINGTON, DC,US;, pages 715 - 718, XP002043738 *
L.C. MATTHEAKIS ET AL.: "An in vitro polysome display system for identifying ligands from very large peptide libraries", PROC. NATL. ACAD. SCI., vol. 91, September 1994 (1994-09-01), NATL. ACAD. SCI.,WASHINGTON,DC,US;, pages 9022 - 9026, XP002043737 *
LEVENS D ET AL: "NOVEL METHOD FOR IDENTIFYING SEQUENCE-SPECIFIC DNA-BINDING PROTEINS", MOLECULAR AND CELLULAR BIOLOGY, vol. 5, no. 9, 1 September 1985 (1985-09-01), pages 2307 - 2315, XP000562760 *
LU Z ET AL: "EXPRESSION OF THIOREDOXIN RANDOM PEPTIDE LIBRARIES ON THE ESCHERICHIA COLI CELL SURFACE AS FUNCTIONAL FUSIONS TO FLAGELLIN: A SYSTEM DESIGNED FOR EXPLORING PROTEIN-PROTEIN INTERACTIONS", BIO/TECHNOLOGY, vol. 13, April 1995 (1995-04-01), pages 366 - 372, XP002033346 *
M.G. CULL ET AL.: "Screening for receptor ligands using large libraries of peptides linked to the C terminus of the lac repressor", PROC. NATL. ACAD. SCI., vol. 89, no. 5, 1992, NATL. ACAD. SCI.,WASHINGTON,DC,US;, pages 1865 - 1869, XP002043736 *
N. S. VISPO ET AL.: "Hybrid rop-pIII proteins for the display of constrained petides in filamentous phage capsid", ANNALES DE BIOLOGIE CLINIQUE, vol. 50, 1993, ELSEVIER, PARIS, FR, pages 917 - 922, XP002043759 *
SCHATZ P J: "CONSTRUCTION AND SCREENING OF BIOLOGICAL PEPTIDE LIBRARIES", CURRENT OPINION IN BIOTECHNOLOGY, vol. 5, no. 5, 1 January 1994 (1994-01-01), pages 487 - 494, XP000564300 *
SCHATZ P J: "USE OF PEPTIDE LIBRARIES TO MAP THE SUBSTRATE SPECIFICITY OF A PEPTIDE-MODIFYING ENZYME: A 13 RESIDUE CONSENSUS PEPTIDE SPECIFIES BIOTINYLATION IN ESCHERICHIA COLI", BIO/TECHNOLOGY, vol. 11, October 1993 (1993-10-01), pages 1138 - 1143, XP000606894 *
T.J. GIBSON ET AL.: "PH domain: the first anniversary", TRENDS IN BIOCHEMICAL SCIENCES, vol. 19, no. 9, September 1994 (1994-09-01), ELSEVIER, AMSTERDAM, NL, pages 349 - 353, XP002043739 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6596485B2 (en) 1998-10-08 2003-07-22 Rigel Pharmaceuticals, Inc. Green fluorescent protein fusions with random peptides
WO2000020574A3 (fr) * 1998-10-08 2000-09-21 Rigel Pharmaceuticals Inc Fusion de proteines d'echafaudage avec des banques de peptides aleatoires
US6180343B1 (en) 1998-10-08 2001-01-30 Rigel Pharmaceuticals, Inc. Green fluorescent protein fusions with random peptides
US6548632B1 (en) 1998-10-08 2003-04-15 Rigel Pharmaceuticals, Inc. Fusions of scaffold proteins with random peptide libraries
US6548249B1 (en) 1998-10-08 2003-04-15 Rigel Pharmaceuticals, Inc. Fusions of scaffold proteins with random peptide libraries
US6562617B1 (en) 1998-10-08 2003-05-13 Rigel Pharmaceuticals, Inc. Fusions of scaffold proteins with random peptide libraries
WO2000020574A2 (fr) * 1998-10-08 2000-04-13 Rigel Pharmaceuticals, Inc. Fusion de proteines d'echafaudage avec des banques de peptides aleatoires
US6936421B2 (en) 1998-10-08 2005-08-30 Rigel Pharmaceuticals, Inc. Structurally biased random peptide libraries based on different scaffolds
US7297482B2 (en) 1998-10-08 2007-11-20 Rigel Pharmaceuticals, Inc. Structurally biased random peptide libraries based on different scaffolds
EP2382990A1 (fr) 2003-04-30 2011-11-02 Universität Zürich Procédés permettant de traiter le cancer à l'aide d'une immunotoxine
US9328336B2 (en) 2012-10-16 2016-05-03 Exxonmobil Research And Engineering Company DGAT genes and methods of use for triglyceride production in recombinant microorganisms
WO2014088583A1 (fr) * 2012-12-06 2014-06-12 Exxonmobil Research And Engineering Company Gènes dgat comprenant des domaines d'homologie de pleckstrine et utilisation dans des micro-organismes recombinants
US8835149B2 (en) 2012-12-06 2014-09-16 Exxonmobil Research And Engineering Company DGAT genes comprising pleckstrin homology domains and methods of use for triglyceride production in recombinant microorganisms

Also Published As

Publication number Publication date
EP0912728A1 (fr) 1999-05-06
JP2000511050A (ja) 2000-08-29

Similar Documents

Publication Publication Date Title
CN108753824B (zh) 用于治疗视网膜营养不良的病毒载体
US6706484B1 (en) Protein/(poly)peptide libraries
KR101778174B1 (ko) 프로테아제 스크리닝 방법 및 이에 의해 확인된 프로테아제
KR102528337B1 (ko) 정의된 서열 및 길이의 dna 단일 가닥 분자의 확장 가능한 생명공학적 생산
AU3204595A (en) Method for selecting high-expressing host cells
US20230043046A1 (en) Recombinant CDKL5 Proteins, Gene Therapy and Production Methods
DK2468861T3 (en) A method for constructing carrier for gene transport
CN110785179A (zh) Wiskott-Aldrich综合征和X连锁血小板减少症中的治疗性基因组编辑
EP0912728A1 (fr) Nouvelles matrices structurelles proteiques synthetiques pour la generation, le criblage et l'evolution de surfaces moleculaires fonctionnelles
US6558924B1 (en) Recombinant expression of insulin C-peptide
CN108913692B (zh) 特异性靶向SATB1基因的sgRNA及其在转录激活中的应用
CN110511950A (zh) Pab蛋白在构建具有类伴侣样蛋白作用的融合蛋白表达载体中的应用
CN110172101A (zh) His-AGR2-DsRed融合蛋白及其制备方法和应用
WO2003062420A1 (fr) Banques de molecules
CN112245578B (zh) 一种covid-19病毒预防性疫苗及其制备方法
US20040191869A1 (en) Crystallography methods
CN109316464B (zh) 包含胰岛样细胞团的制剂
CN114686411B (zh) 一种工程菌的混合物、用于感应和吸附重金属污染物的试剂盒和处理方法
CN109913484A (zh) 一种双向表达t载体以及其制备方法和应用
KR100566091B1 (ko) 이황화결합 가교로 형성된 키메라 재조합접착도메인-작용기 융합체의 이량체 및 이의 제조 방법
CN109336982B (zh) 基因改造的干细胞及其应用
KR102286791B1 (ko) SrbA 단백질 단편과 골지 상주 단백질이 융합된 단백질을 발현하는 재조합 균주 및 이의 응용
CN112575020B (zh) 能生物合成甘油葡萄糖苷且耐盐的聚球藻基因工程菌及构建方法
KR102149088B1 (ko) SrbA 단백질 단편과 골지 상주 단백질이 융합된 단백질을 발현하는 재조합 균주 및 이의 응용
CN110498859A (zh) 重组PLB-hbFGF融合蛋白及其应用

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): JP US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 1997927094

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1997927094

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1997927094

Country of ref document: EP