WO2000036093A2 - Intein-mediated cyclization of peptides - Google Patents
Intein-mediated cyclization of peptides Download PDFInfo
- Publication number
- WO2000036093A2 WO2000036093A2 PCT/US1999/030162 US9930162W WO0036093A2 WO 2000036093 A2 WO2000036093 A2 WO 2000036093A2 US 9930162 W US9930162 W US 9930162W WO 0036093 A2 WO0036093 A2 WO 0036093A2
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- Prior art keywords
- peptide
- split intein
- host system
- molecule
- nucleic acid
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- UAEPNZWRGJTJPN-UHFFFAOYSA-N CC1CCCCC1 Chemical compound CC1CCCCC1 UAEPNZWRGJTJPN-UHFFFAOYSA-N 0.000 description 1
Classifications
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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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/50—Cyclic peptides containing at least one abnormal peptide link
- C07K7/52—Cyclic peptides containing at least one abnormal peptide link with only normal peptide links in the ring
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/64—Cyclic peptides containing only normal peptide links
-
- 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
- C12N15/1055—Protein x Protein interaction, e.g. two hybrid selection
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
Definitions
- the invention relates to the field of biochemistry. More particularly, the invention
- Ring closure, or cyclization can reduce the rate at which peptides are degraded in
- methods involve first expressing linear precursors of cyclic peptides in cells (e.g., bacteria) to produce linear precursors of cyclic peptides and then adding of an exogenous agent such as a
- protease or a nucleophilic reagent to chemically convert these linear precursors into cyclic
- cyclic peptides can be screened for pharmacological activity.
- a library containing large numbers of different cyclic peptides can be prepared and
- the library is mixed with the target ligand, and those members of the library that bind to the
- target ligand can be isolated and identified by amino acid sequencing. Similarly, libraries of
- cyclic peptides can be added to assays for a specific biological activity. Those cyclic peptides
- screening assays can prove laborious and time-consuming. For instance, screening
- the cyclic peptides in that area of the library can then be isolated and sequenced.
- library of linear peptides can be added to a screening assay, and those bacteria that express the
- screened for characteristic can be picked directly from the assay.
- the picked bacteria can be picked directly from the assay.
- the picked bacteria can be picked directly from the assay.
- NRPS non-ribosomal peptide synthetase
- NRPS entails cloning the subunit(s) which incorporate the desired monomers and introducing
- NRPS subunits to host cells and devising a method for ensuring that the subunits assemble into the correct supramolecular structures.
- nucleic acid molecule is constructed such that a
- nucleotide sequence encoding the peptide to be cyclized is flanked on one end with a
- nucleotide sequence encoding the carboxy-terminal portion of a split (or trans) intein (C-
- intein or I c a nucleotide sequence encoding the amino-terminal portion of a split intein (N-intein or I N ).
- split intein components i.e., I c and I n
- I c and I n split intein components
- the invention features a non-naturally occurring nucleic acid molecule
- intein intermediate a thioester intermediate, or a lariat intermediate.
- Both the first portion of a split intein and the second portion of a split intein can be
- both of split intein portions can be derived from non-naturally occurring split inteins such as
- the invention features a non-naturally occurring nucleic acid
- first target peptide is interposed between the first portion of a split intein and the
- the first portion of a split intein can be complementary to the third portion of a split intein but not complementary to the
- second portion of a split intein, and the second portion of a split intein can be complementary
- an expression vector comprising a nucleic acid molecule
- the expression vector of the invention can also contain a regulatory sequence that facilitates
- the nucleic acid molecule of the vector can be any nucleic acid molecule of the vector.
- nucleotide sequence that encodes a peptide that facilitates screening of the cyclized
- the expression vector can also be inducible.
- the invention features an expression vector encoding a polypeptide
- Expression vectors of the invention can be a
- plasmid a bacteriophage, a virus, a linear nucleic acid molecule, or other type of vector.
- the invention additionally features a substantially pure polypeptide having a first
- polypeptide can be one that spontaneously splices in the host system to yield a cyclized form
- the target peptide or it can be a splicing intermediate.
- the host system harboring a nucleic acid molecule of the invention.
- the host system can be a prokaryote such as a bacterium, an archaebacterium, a
- eukaryote such as a yeast or a mammalian cell, a plant cell, an in vitro
- the invention features a method for making a peptide molecule.
- This method includes the steps of: providing an isolated nucleic acid molecule of the
- method can also feature the step of purifying the cyclized form of the target peptide from the
- the step of expressing the isolated nucleic acid in another variation of this method, the step of expressing the isolated nucleic acid
- This method can also feature the step of purifying the splicing intermediate of a cyclized form of the target peptide from the host system. Yet another variation of this method,
- the target peptide is produced in a cyclized form in
- Another aspect of the invention is a method of preparing a library of peptide
- This method involves the steps of providing a plurality of nucleic acid molecules
- the plurality of nucleic acid molecules is interposed between a nucleic acid molecule encoding a first portion of a split intein and a nucleic acid molecule encoding an second portion of a split intein in each of the formed expression vectors such that expression of the
- the invention includes a method of screening a peptide
- This method includes the steps of: providing a
- nucleic acid molecule that encodes a polypeptide comprising a first portion of a split intein, a
- split intein and the second portion of a split intein providing the host system; introducing the
- polypeptide in the host system or a splicing intermediate of a cyclized form of the target
- the predetermined characteristic includes the ability to
- predetermined characteristic includes the steps of (a) contacting the peptide molecule to the target molecule and (b) determining whether the peptide molecule binds to the target
- the predetermined characteristic is the ability to modulate a
- characteristic comprises the steps of (a) contacting the peptide molecule to a system
- binds to a target molecule or modulates a biochemical reaction can measured by observing a
- the target molecule in these methods can be a cell-associated molecule such as a
- membrane-associated molecule e.g., a nuclear molecule or one
- organelles such as mitochondria, lysosomes, endoplasmic reticula, chloroplasts,
- golgi golgi, and periplasm). It can also be an extracellular molecule.
- the biochemical reaction can be a cell associated-process such as an intracellular
- metabolic event a membrane-associated event, a nuclear event. It can also be an extracellular
- the invention also features a method for purifying a cyclic peptide from a mixture.
- This method includes the steps of: providing a mixture containing a splicing intermediate
- phase support having a ligand thereon that specifically binds the affinity tag such that the
- support becomes specifically bound with the splicing intermediate; washing the support to remove non-specifically bound matter from the support; adding to the support a reagent that
- the invention also includes a method for purifying a
- cyclic peptide from a mixture that includes the steps of: providing a mixture containing a splicing intermediate conjugated with an affinity tag; mixing the conjugated splicing
- affinity tag such that the support becomes specifically bound with the splicing intermediate
- This method includes the steps of: providing a solid phase support having the splicing intermediate specifically bound thereon; contacting
- non-naturally occurring means being directly or
- nucleic acid molecule is one that has been produced through human manipulation, and not
- nucleic acid molecule any chain of two or more nucleotides
- a nucleic acid molecule can be a DNA or an RNA.
- peptide means a chain of two or more amino acids bonded
- polypeptide in sequence, and includes polypeptides and proteins.
- polypeptide is meant a polymer
- protein any chain of amino acids and includes peptides, polypeptides, proteins,
- modified proteins such as glycoproteins, lipoproteins, phosphoproteins, metalloproteins, and the like.
- linear peptide is a peptide that is not in a circular form, and generally has both a carboxy-terminal amino acid with a free carboxy-terminus and an amino-terminal amino acid
- cyclic peptide is a peptide that has been “cyclized.”
- cyclic means having constituent atoms forming a ring.
- cyclize means to make the peptide into a cyclic or "cyclized” form.
- a linear peptide is "cyclized" when its free amino-terminus is covalently bonded to
- a “splicing intermediate” is a polypeptide generated during the intein-
- Splicing intermediates include "active-intein intermediates" (i.e., those with
- target peptide a peptide to be cyclized or displayed in a
- splicing intermediate For example, a peptide interposed between a carboxy-terminal portion
- intein means a naturally-occurring or artificially-
- splicing reaction during post-translation processing of the protein.
- a list of known inteins is published at http://www.neb.com/inteins.html.
- a "split intein” is an intein that has two or
- the word "interposed" means placed in between.
- polypeptide having a first peptide interposed between a second and a third peptide, the chain
- a plurality of peptides having "heterogenous amino acid sequences" means that the
- plurality of peptides is composed of at least two, but generally a large number of, different peptides of disparate amino acid sequence.
- host system refers to any medium or vehicle in which a
- nucleic acid molecule can be transcribed, replicated, and/or translated; and/or any medium or
- a precursor polypeptide within a host is added to the addition of an exogenous substance.
- a precursor polypeptide within a host is added to the addition of an exogenous substance.
- splice or "splices” means to excise a central portion of the
- polypeptide to form two or more smaller polypeptide molecules.
- splicing also occurs
- polypeptide As used herein, the word "derived" means directly or indirectly obtained from,
- expression vector means a vehicle that facilitates
- vector is "inducible" when adding an exogenous substance to a host system containing the
- expression vector causes the vector to be expressed (e.g., causes a nucleic acid molecule within the vector to be transcribed into mRNA).
- regulatory sequence means a nucleotide sequence which
- promoters and enhancers are regulatory sequences.
- a first peptide is
- an “isolated” or “substantially pure” substance is one that has been
- polypeptide is
- a "progenitor DNA” is particular deoxyribonucleic acid from which mutations are made or based upon.
- target molecule any molecule used to determine the binding
- binding means that one molecule recognizes and adheres to another
- One molecule "specifically binds" another molecule if it has a binding affinity
- a "cell-associated process” is one that takes place within a cell or in the near vicinity
- a "membrane-associated event” is a cell-associated process that takes place on the
- a "nuclear event” is a cell-associated process that takes place in the nucleus of a cell.
- an "extracellular reaction” is one that does
- hybrid system two-hybrid systems, reverse two-hybrid
- Figure 1 is a schematic illustration of an overview of a general cyclization reaction
- Figure 2 is a schematic illustration of a series of chemical reaction steps that occur in
- Figure 3 is a genetic map of (a) plasmid pARCP, (b) plasmid pARCP-DHFR, (c)
- plasmid pARCPAH-DHFR (d) a modified vector having a cysteine (TGY) or serine (TCN)
- N represents any nucleobase
- S represents C
- G and Y represents pyrimidines), ( e) plasmid pARCP-p, and (f) plasmid pARCBD-p.
- Figure 4 is a photograph of a sodium dodecylsulfate polyacrylamide gel electro-
- Figure 5 is a graph of DHFR activity of wild-type (triangles) and cyclic DHFR
- Figure 6 is a schematic illustration of the expected endoproteinase Lys-C digestion
- Figure 7 is a photograph of FeCuY plates used in an in vivo assay to detect tyrosinase
- Figure 8 is a schematic illustration of a method for purifying cyclic peptides within the
- Figure 9 is a schematic illustration of another method for purifying cyclic peptides
- Figure 10 is a schematic illustration of a solid phase support/affinity chromatography-
- FIG 11 is a schematic illustration of another solid phase support/affinity
- Figure 12 is a schematic illustration of another solid phase support/affinity
- Figure 13 is a schematic illustration of another solid phase support/affinity
- Figure 14 is a schematic illustration of the use of aptamer scaffolds in the invention.
- Figure 15 is a schematic illustration of two reactions for preparing aptamers within the invention.
- Figure 16 is a schematic illustration of a method for screening within the invention.
- Figure 17 is a schematic illustration of another method for screening within the invention.
- Figure 18 is a schematic illustration of another method for screening within the invention.
- Figure 19 is a schematic illustration of another method for screening within the
- the split intein physically come together to form an active intein in a conformation that also
- peptide is stabilized such that heteroatom from the other portion of the intein (e.g., I c ) can
- backbone cyclic peptide product i.e., the lactam form.
- nucleic acid molecules in a loop configuration can be produced.
- cyclic peptides or splicing intermediates can be prepared to generate a library of cyclized or partially-cyclized peptides that can be screened for particular characteristics.
- an overview of an embodiment of the invention includes a
- a split intein e.g., I c
- a peptide to be cyclized i.e., a
- the nucleic acid molecule can be any suitable nucleic acid molecule.
- a split intein e.g., I N .
- the nucleic acid molecule can be any suitable nucleic acid molecule.
- the nucleic acid can be transcribed and translated into a precursor polypeptide having the peptide to be cyclized interposed between the two split intein portions.
- Nucleic acids molecules within the invention include those that encode a polypeptide
- expression of the nucleic acid molecule in a host system in one embodiment, expression of the nucleic acid molecule in a host system
- nucleic acids of the invention can be prepared according to
- nucleic acid molecule within
- the invention can be made by separately preparing a polynucleotide encoding the first portion
- polynucleotide encoding the target peptide.
- the three polynucleotides can be ligated together
- nucleic acid molecule that encodes a polypeptide having the target peptide
- inteins portion of a split intein of the nucleic acid molecules within the invention can be derived from known inteins. A fairly comprehensive and descriptive list of such inteins is published
- inteins can be used as long as they are compatible with invention.
- split inteins can be used to generate the intein portions of nucleic acid molecules within the
- nucleic acid The nucleic acid
- split intein One example of a naturally-occurring split intein that may be used is Ssp DnaE (Wu et al,
- chain of amino acids can be artificially split using known techniques. For example, two or more nucleic acid molecules encoding different portions of such inteins can be made so that
- nucleic acids that encode such non-naturally occurring intein components can be
- nucleic acid molecules include Psp Pol-1
- GyrA (Evans, T.C. et al, J. Biol. Chem. 274:18359, 1999), and Pfu (Otomo et al,
- random sequence can be prepared chemically by solid phase synthesis using commercially available equipment and reagents. Polymerase chain reaction can also be used to prepare
- restriction endonucleases can be used to enzymatically digest a larger nucleic acid
- nucleic acid molecules of the invention fragments that can be used to prepare nucleic acid molecules of the invention.
- Polynucleotides that encode peptide sequences to be cyclized are preferably prepared
- polynucleotide encodes an asparagine, serine, cysteine, or
- encode peptide sequences for production of splicing intermediates are preferably
- cysteine or threonine residue so that the cyclization reaction is prevented.
- intein portions to a nucleic acid molecule encoding a target peptide (or peptide
- nucleic acid constructs that encode more than one set of two portions of a split intein
- the invention includes nucleic acids molecules
- N an integer greater than or equal to 1
- target peptide i an integer greater than 1 representing the position of an target peptide in the precursor polypeptide
- target peptide 1 is between intein portionl & 2
- target peptide 2 is between intein portions 3 & 4 etc.
- intein portions 2i-l and 2i are not complementary (i.e. able to physically interact to catalyze a splicing event)
- target peptide i can not cyclize. If, however,
- intein portion 2i is complementary with intein portion 2i+l and intein portion 2N is complementary with intein portion 1, the entire ensemble of N polypeptides can perform N-l
- trans splices between 2 polypeptides
- l cis splice ligating the two ends together
- 1-N target peptides are covalently attached to one another in a cyclic peptide/protein (e.g., intein portions 2 & 3 trans-splice target peptides 1 & 2; intein
- trans-splice portions four & five trans-splice target peptides 2 & 3; intein portions 2N-2 & 2N-1 trans- splice target peptides N-l & N; and intein portions N & 1 cis-splice to close the cyclic product containing the N target sequences).
- the order of trans/cis splicing events is irrelevant.
- the slowest splicing species (whether it is the complementary intein portion 2N&1, 2&3 or 80&81) will by default perform the cis-splice.
- nucleic acid constructs can be made that express two or more polypeptides each composed of a target peptide interposed between two portions of a split intein where the
- intein components are not complementary (i.e., do not derive from the same intein or
- polypeptide could be cyclized unless it was expressed in the presence of a second polypeptide
- polypeptide per construct within the invention could encode only one polypeptide per construct or more than one polypeptide per construct (e.g., a bi-functional plasmid).
- the expression vectors of the present invention can be prepared by inserting polynucleotides encoding a target peptide into any suitable expression vector that can facilitate expression of the polynucleotide in a host system.
- suitable vectors include
- type of host system selected e.g., in vitro systems, prokaryotic cells such as bacteria, and
- eukaryotic cells such as yeast or mammalian cells
- expression conditions selected.
- Expression vectors within the invention can include a stretch of nucleotides that
- the regulatory domain can be a promoter or an enhancer.
- Expression vectors within the invention can include nucleotide sequences that encode
- an affinity tag such as a chitin-binding
- a host system e.g., an
- affinity tag such as a chitin-binding domain, a biotin tag, a colored or light-emitting label; a
- the expression vectors within the invention are produced
- an expression vector of the invention can be an
- inducible expression vector such as an arabinose inducible vector.
- arabinose inducible vector Such vectors can be
- vectors can be selected for use in the invention based on their compatibility with known bacterial expression strains and hybrid systems. See, e.g., Zhang et al, Curr.
- Polypeptides within the invention include any that can be produced by expression of a
- nucleic acid of the invention for example, a substantially pure precursor polypeptide that has a target peptide (or a peptide to be displayed by a splicing intermediate) interposed
- the target peptide may be any suitable polypeptide.
- the target peptide may be any suitable polypeptide.
- these are produced by splicing of a precursor
- polypeptide of the invention can be of any amino acid
- the splicing intermediate can be any sequence that can be cyclized by the methods of the invention.
- Hosts systems that may be used in the invention include any systems that support transcription, translation, and/or replication of a nucleic acid molecule of the invention
- the host system can take the form of an in vitro transcription/translation system.
- Such systems can be fabricated in the laboratory according to published techniques or can be
- STP2-T7 (cat. No. 69950-3) and STP-SP6 (cat. No. 69997-3) are available from Novagen (Madison, Wl). Promega (Madison, Wl) also sells such
- Non-living host systems for use in the invention can also be derived from a living organism.
- a cell lysate such as a reticulocyte lysate can be
- Host systems can also take the form of living organisms.
- Living organisms are
- prokaryotes such as bacteria (e.g., Escherichia coli) and eukaryotes such as yeasts and
- mammalian e.g., human, murine, bovine, ovine, porcine, etc.
- Archaebacteria plant
- a suitable host system for a particular application based on known applications of the different host systems. For example, where large scale production of a cyclic peptide is desired, a
- polypeptides of the invention can be prepared by conventional methods of
- polypeptides within a known amino acid sequence For example, polypeptides within
- the invention can be made by solid phase synthesis using commercially available equipment
- polypeptides of the invention are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids.
- polypeptides of the invention are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino acids that are amino
- nucleic acid molecules within the invention can be incorporated into an
- a preferred method for making a cyclic peptide or a splicing intermediate includes the
- target peptide interposed between the first portion of a split intein and the second portion of a
- nucleic acid molecule in the host system produces the peptide molecule in the form of a
- production of the polypeptides, cyclic peptides, or splicing intermediates takes place in vivo (e.g., with a living host system) and in
- peptide e.g., a protease or a thiol
- Production of polypeptides, cyclic peptides, or splicing intermediates can be
- Exemplary techniques that can be used include conventional chromatography, HPLC,
- electrophoresis such as sodium dodecyl sulfate polyacrylamide gel
- thermostability assays etc.
- the invention also includes a
- an affinity tag for purifying a cyclic peptide from a mixture.
- an affinity tag for purifying a cyclic peptide from a mixture.
- This method includes the steps of:
- the affinity tag can any molecule that can bind a ligand on a solid phase support.
- the affinity tag can be a chitin-binding domain where the ligand
- ligand is chitin (see examples section below) or it can be a biotin tag where the ligand is
- streptavidin Many other affinity tag-ligand pairs are known and can be used in the invention.
- the affinity tag specifically binds the ligand on the solid phase support, the cyclic
- the support can be any suitable support.
- the support can be any suitable support.
- a buffer e.g. a high salt, acid or alkaline buffer
- peptide can then be eluted from the solid phase support using a buffer containing a substance that separates the tag from the ligand (e.g., a competitive inhibitor such as excess
- unconjugated affinity tag or a denaturing agent
- an enzyme or chemical reactant that
- Cyclic peptides can also be purified from a mixture using splicing intermediates.
- a method for purifying a cyclic peptide from a mixture includes the steps of: (a)
- steps (d) and (e) are reversed so that step (d) is eluting the splicing intermediate
- step (e) is adding to the eluted splicing intermediate a reagent that
- cyclic peptide from a splicing intermediate include thiols, proteases, and other substances which can catalyze cyclization of the splicing intermediate.
- a cyclic ester can be immobilized on an affinity column.
- the resulting cyclic peptide column can be used for the affinity purification of the cyclic
- proteolytic methods can be employed to liberate the cyclic ester from the affinity tag and I c depending upon the sequence of the cyclic peptide product.
- FIG. 8 a method for purifying cyclic peptides is shown.
- an active intein intermediate (specie 1) is mutagenized to replace the catalytic
- step A with a non-catalytic amino acid (Y) and to introduce an affinity tag
- step C This molecule is then passed through
- step D having a solid phase support with a ligand thereon that
- step F liberates the lactone intermediate (specie 5).
- step G yields the thermodynamically preferred amide cyclic product (specie 6).
- step A replaces the catalytic asparagine (step A) with a non-catalytic amino acid (Y) and to introduce
- step D having a solid phase support with a ligand thereon
- step E Separation of the affinity tag (step E) from the ligand (e.g., using a molecule that competitively inhibits the tag-ligand
- protease to cleave the tag allows recovery of the lariat intermediate (specie 4).
- step F chemical cleavage at amino acid Y (step F) liberates the lactone intermediate (specie 5).
- step G yields the thermodynamically preferred amide cyclic
- a method of preparing a library of cyclic peptides and/or splicing intermediates includes the steps of: (a) providing a plurality of nucleic acid
- nucleic acid molecule encoding a first portion of a split intein and a nucleic acid molecule encoding an second portion of a split intein in each of the formed
- target peptides and (c) expressing the expression vectors in the host system.
- polynucleotides encoding peptides with random sequences that can be circularized or
- sequences can be incorporated as the target peptide (or the peptide to be displayed in a
- peptide in a combinatorial peptide library binds specifically to a target protein
- a target peptide interposed between a first portion of a split intein and a second portion of a
- Step (a) can be performed as described elsewhere herein by, for example, using
- the resulting three polynucleotides can then be fused (e.g., ligated) together to
- the host system can be any of those described
- nucleic acid molecule e.g., a bacterium, a yeast, a
- the nucleic acid can be introduced into the host system by known means
- the nucleic acid molecules can be introduced into a cell by electroporation
- bacteriophage vector when host system is a bacterium, using a plasmid construct, using a
- the host system can be placed under conditions that cause the peptide molecule to be
- this step can include adding
- affinity chromatography to identify molecules which specifically bind cyclic peptides or splicing intermediates; using phage display technology; and using aptamer peptide fusion
- Cyclic peptides or splicing intermediates can be immobilized on a solid phase support
- step 1 is mutagenized to replace the catalytic asparagine (step A) with a non-catalytic amino acid (Y) and to introduce an affinity tag upstream of I c (step B) to yield
- step C This molecule is then passed though an affinity column (step D) having a
- target molecules can be removed from the column and biochemically analyzed (e.g.,
- non-catalytic amino acid Y
- step B to introduce an affinity tag downstream of I N
- step C This molecule is then passed though an affinity column (step C).
- step E allows recovery of the lariat intermediate (specie 4).
- target molecules i.e., candidates for binding the splicing intermediates
- step 1 is mutagenized to replace the I N nucleophile (step A) with a non- catalytic amino acid (Z) and to introduce an affinity tag downstream of I N (step B) to yield an
- step C This protein is then passed though an affinity column
- step D having a solid phase support with a ligand thereon that specifically binds the affinity
- target molecules i.e., candidates for binding the splicing intermediates
- intein intermediate (specie 1) is mutagenized to replace the I N nucleophile (step A) with a
- non-catalytic amino acid (Z) and to introduce an affinity tag upstream of I c (step B) to yield
- step D having a solid phase support with a ligand thereon that specifically binds the affinity
- target molecules i.e., candidates for binding the splicing intermediates
- Methods of screening molecules using phage display are also within the invention.
- libraries comprising phage particles displaying loop target peptides can be prepared
- a target molecule for instance, a target molecule
- the amino acid sequences of the looped peptides can be any amino acid sequences of the looped peptides.
- Peptides aptamers are polypeptides that contain a conformationally constrained target
- splicing intermediates can function as aptamers, known methods of analyzing aptamers can
- cyclic proteins can be used as aptamer scaffolds in
- an aptamer library can be expressed as an I c -scaffold-I N fusion protein (specie 1). Procession
- step A of the intein-mediated cyclization reaction in vivo (step A) yields an I N , an I c , and a cyclic scaffold protein (specie 2).
- the aptamer library is displayed in the linker region between the
- N represents any amino acid and the subscripts n and
- n are any integral number equal or greater than 0, and X represents serine, threonine, or
- cysteine Other examples of using aptamers are also within the invention. Referring to FIG. 15,
- reaction I an active intein intermediate is mutagenized to
- step A replace the nucleophilic amino acid from I N (step A) with a non-catalytic amino acid (Z).
- reaction II an active intein intermediate is mutagenized to replace the catalytic asparagine
- step A with a non-catalytic amino acid (Y) to yield specie 2. Progression of the intein-
- step B mediated cyclization reaction proceeds in vivo (step B) and arrests at the lariat intermediate
- stage (specie 3) allowing members of a peptide library to be displayed as a constrained
- the yeast two-hybrid system is a well-studied method for analyzing in vivo protein-
- an active intein interacts with a splicing intermediate is within the invention.
- an active intein interacts with a splicing intermediate.
- non-catalytic amino acid Y
- I N DNA-binding domain downstream of I N
- step C I N and I c form a strong non-covalent complex.
- the resulting lariat intermediate is then co-expressed with a target protein attached to a DNA- binding domain (step D). Interaction of the lariat intermediate with the target protein (specie
- step E causes activation of a promoter region (step E) leading to expression of the reporter gene
- This method can be modified such that a known molecule (in place of an unknown target protein) is attached to a DNA-binding domain, so that lariat intermediates displaying a looped
- step A 1) is mutagenized to replace the catalytic asparagine (step A) with a non-catalytic amino acid
- step C This molecule is then co-expressed with a target protein attached to a DNA-
- step D Interaction of the lariat intermediate with the target protein (specie 4) causes activation of a promoter region (step E) leading to expression of the reporter gene
- This method can be modified such that a known molecule (in place of an unknown target
- step A 1) is mutagenized to replace the I N nucleophile (step A) with a non-catalytic amino acid (Z)
- step C Interaction of the fusion protein with the target protein (specie 3)
- step D causes activation of a promoter region (step D) leading to expression of the reporter gene (*).
- This method allows identification of target molecules able to bind the fusion protein .
- an active intein intermediate (specie 1) is mutagenized to replace the I N nucleophile (step A) with a non-catalytic amino acid (Z)
- This molecule is then co-expressed with a target protein attached to a DNA-binding
- step C Interaction of the fusion protein with the target protein (specie 3) in step D causes activation of a promoter region (step E) leading to expression of the reporter gene (*).
- This method allows identification of target molecules able to bind the fusion protein .
- cyclic peptides and splicing intermediates within the invention can be specifically described
- peptides can be attached to the cyclic peptides or splicing intermediates of the invention to
- intermediates of the invention can be targeted to cellular locales such as mitochondria,
- lysosomes endoplasmic reticula, chloroplasts, golgi, periplasm, the nucleus, the plasma
- This method for targeting can also be used in the methods for generating a
- the gene for the Ssp DnaE N-intein (I N ) was amplified from Ssp 6803 genomic DNA
- the Ssp DnaE I c gene was amplified similarly with primers introducing 5'-Nc ⁇ l and 3'-Nde ⁇
- Plasmid pDIMCP resulted from individually cloning the intein fragments into pDIMC7 [identical to pDIMC ⁇ (see Ostermeier et al., Proc. ⁇ atl. Acad. Sci.
- primers introducing a 5'-Ndel site followed by (CAC) 6 (encoding six histidine residues) and a 3'-Rs * tI site, digested with Ndel/Pstl and ligated into NdellNsil digested pARCP or pARCPAH
- Plasmid pARCP-p was produced in three steps from pDIMCPAH: (1) Quick-Change mutagenesis introduced an AfH site into I ⁇ , generating
- Thermostability was assayed by preincubation of 100 nM wild type or cyclic DHFR at
- Tris tris(hydroxymethyl)aminomethane
- ethanolamine 25 mM ethanolamine
- NaCl 100 mM NaCl
- NADPH reduced nicotinamide adenine dinucleotide
- E. coli strains XLl-Blue, DH5a or BL21-D ⁇ 3 harboring pARCP-p were grown and
- the tyrosinase gene (including ORF 438) from Streptomyces antibioticus (Bernan et al, Gene 37:101, 1985) was amplified with Vent -polymerase from pU702 (ATCC no. 35287) with primers introducing 5' N ⁇ fel and 3' EeoRI restriction sites.
- the PCR product was amplified with Vent -polymerase from pU702 (ATCC no. 35287) with primers introducing 5' N ⁇ fel and 3' EeoRI restriction sites.
- the PCR product was
- the genes encoding Ssp I c and I N were amplified from Ssp genomic DNA by standard molecular biology methods (Sambrook, et al, supra) and serially ligated into pDIMC7.
- the genes encoding Ssp I c and I N were amplified from Ssp genomic DNA by standard molecular biology methods (Sambrook, et al, supra) and serially ligated into pDIMC7.
- CP cyclization precursor
- DHFR gene was cloned between the Ndel and Nsil sites of pARCP to create an in frame
- DHFR also introduced a sequence encoding a six-histidine tag at the 5' end of the DHFR gene
- Plasmid pARCP-DHFR (b in Fig. 3) encodes wild type I c , which has an alanine residue neighboring the terminal asparagine. Plasmid
- pARCPAH-DHFR ( c in Fig 3) incorporates an alanine-to-histidine mutation at the
- pseudostellarin F cyclo-[SGGYLPPL]
- the vector was modified by silent mutation to create an Aflll site at the 5'-end of the I N gene (d in
- vector produced plasmid pARCP-p (e in Fig. 3).
- a Kpnl site was introduced at the 3'-end of the I N gene in order to fuse the gene for the chitin-binding domain to the pseudostellarin-
- Lane 3 arabinose induced XLIBlue/pARCPAH-DHFR. Lane 4: lane 3 crude lysate after methotrexate agarose. Lane 5: lane 4 material post FPLC.
- Lane 6 Wild-type DHFR.
- Cyclic DHFR had steady-state kinetic parameters and substrate, cofactor and
- methotrexate dissociation constants which were indistinguishable from the wild type enzyme
- purified protein was cyclic DHFR. Digestion of the wild type enzyme produces amino-
- purified material was resistant to proteolysis compared to the wild type enzyme, and mass
- Pseudostellarin F production was readily detected in vivo through inhibition of
- FIG. 7 recombinant Streptomyces antibioticus tyrosinase
- XLI-Blue cells were co-transformed with pDIM-NY and either pARCP2-6H (a & b) or pARCP-p ( c & d).
- the cells were plated on FeCuY plates with chloramphenicol (50 ug/ml),
- a chitin-binding domain was fused to the carboxy-terminus of I N to affinity-purify
- Pseudostellarin F was recovered from the unretained material by 1 -butanol
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Abstract
Description
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Priority Applications (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE69930164T DE69930164T2 (en) | 1998-12-18 | 1999-12-18 | INTEIN-MEDIATED CYCLISATION OF PEPTIDES |
JP2000588342A JP4544601B2 (en) | 1998-12-18 | 1999-12-18 | Cyclic peptide |
EP99966402A EP1141250B1 (en) | 1998-12-18 | 1999-12-18 | Intein-mediated cyclization of peptides |
AU21948/00A AU781215B2 (en) | 1998-12-18 | 1999-12-18 | Cyclic peptides |
CA002355934A CA2355934A1 (en) | 1998-12-18 | 1999-12-18 | Intein mediated cyclization of peptides |
US09/868,469 US7354756B1 (en) | 1998-12-18 | 1999-12-18 | Intein-mediated cyclization of peptides |
US11/670,112 US7846710B2 (en) | 1998-12-18 | 2007-02-01 | Cell comprising an intein for the production of a cyclic peptide |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US11272398P | 1998-12-18 | 1998-12-18 | |
US60/112,723 | 1998-12-18 | ||
US15807799P | 1999-10-07 | 1999-10-07 | |
US60/158,077 | 1999-10-07 |
Related Child Applications (2)
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US09/868,469 A-371-Of-International US7354756B1 (en) | 1998-12-18 | 1999-12-18 | Intein-mediated cyclization of peptides |
US11/670,112 Continuation US7846710B2 (en) | 1998-12-18 | 2007-02-01 | Cell comprising an intein for the production of a cyclic peptide |
Publications (3)
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WO2000036093A2 true WO2000036093A2 (en) | 2000-06-22 |
WO2000036093A3 WO2000036093A3 (en) | 2000-10-19 |
WO2000036093A9 WO2000036093A9 (en) | 2001-08-16 |
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PCT/US1999/030162 WO2000036093A2 (en) | 1998-12-18 | 1999-12-18 | Intein-mediated cyclization of peptides |
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US (2) | US7354756B1 (en) |
EP (1) | EP1141250B1 (en) |
JP (1) | JP4544601B2 (en) |
CN (1) | CN1179040C (en) |
AT (1) | ATE318894T1 (en) |
AU (1) | AU781215B2 (en) |
CA (1) | CA2355934A1 (en) |
DE (1) | DE69930164T2 (en) |
DK (1) | DK1141250T3 (en) |
ES (1) | ES2260956T3 (en) |
WO (1) | WO2000036093A2 (en) |
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Publication number | Publication date |
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EP1141250A2 (en) | 2001-10-10 |
DE69930164T2 (en) | 2006-11-23 |
US7354756B1 (en) | 2008-04-08 |
DE69930164D1 (en) | 2006-04-27 |
AU781215B2 (en) | 2005-05-12 |
WO2000036093A9 (en) | 2001-08-16 |
AU2194800A (en) | 2000-07-03 |
JP2003534768A (en) | 2003-11-25 |
ES2260956T3 (en) | 2006-11-01 |
US7846710B2 (en) | 2010-12-07 |
US20070207502A1 (en) | 2007-09-06 |
CN1354788A (en) | 2002-06-19 |
EP1141250B1 (en) | 2006-03-01 |
ATE318894T1 (en) | 2006-03-15 |
CN1179040C (en) | 2004-12-08 |
CA2355934A1 (en) | 2000-06-22 |
DK1141250T3 (en) | 2006-07-10 |
WO2000036093A3 (en) | 2000-10-19 |
JP4544601B2 (en) | 2010-09-15 |
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