WO2000015789A1 - Analogues recombinants de proteines bioadhesives contenant de l'hydroxyproline - Google Patents

Analogues recombinants de proteines bioadhesives contenant de l'hydroxyproline Download PDF

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Publication number
WO2000015789A1
WO2000015789A1 PCT/US1999/020463 US9920463W WO0015789A1 WO 2000015789 A1 WO2000015789 A1 WO 2000015789A1 US 9920463 W US9920463 W US 9920463W WO 0015789 A1 WO0015789 A1 WO 0015789A1
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Prior art keywords
proline
pro
analog
hyp
precursor protein
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PCT/US1999/020463
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English (en)
Inventor
David N. Paolella
Elliott A. Gruskin
Douglas D. Buechter
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United States Surgical Corporation
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Priority to AU59100/99A priority Critical patent/AU5910099A/en
Publication of WO2000015789A1 publication Critical patent/WO2000015789A1/fr

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    • 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
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates

Definitions

  • Engineered polypeptides having bioadhesive properties in particular, a method for the production of bioadhesive polypeptides wherein hydroxyproline and/or other proline analogs is incorporated into the polypeptide at the translational level, thereby obviating the need for post-translational proline hydroxylation.
  • Marine mussels and other sessile invertebrates such as platyhelminths, annelids, and tunicates secrete adhesive substances in order to affix themselves to underwater objects.
  • Mussels of the genus Mytilus secrete an adhesive precursor substance from their foot that upon cure forms a
  • M. edulis A major component of the adhesive deposited by M. edulis has been identified as a hvdroxylated protein of about 130,000 daltons (Waite, J. H., J. Biol. Chem., Vol. 258, pp. 291 1-2915 ( 1983)).
  • U.S. Pat. No. 4,496,397 to Waite further discloses that the protein from M. edulis comprises a large number (75-80) of tandem repeats
  • Y' is a serine, valine, aspartic acid, or leucine residue and Y 2 is an alanine, proline,
  • protein from the mussel Aulecomya ater comprises a repeating heptapeptide having the
  • Precursor proteins comprising the above sequences have a large number of basic and hydroxylated amino acids, including lysine. serine, threonine, and others.
  • hydroxylated amino acids include
  • amino acids because they are incorporated into the protein as tyrosine and proline during
  • post-translational modification * ' or in this instance, "post-translation hydroxylation " '.
  • Post-translational hydroxylation is believed to be
  • the hydroxyl groups likely chelate with metals or other ions present on the substrates on which mussels anchor.
  • Oxidation of the dihydroxy groups of the tyrosine yield ortho-phenols, which can then crosslink and form or strengthen the adhesive bond. See, Waite, J. H., In Mollusca, Vol. 1, pp.467-504 (1983); Pizzi, A., et al., Ind. Eng. Chem. Prod. Res. Dev., Vol. 21, pp. 309-369
  • Adhesives and sealants in current therapeutic use include both proteinaceous and
  • One proteinaceous formulation is a mixture of the human proteins
  • fibrinogen fibrinogen, thrombin, and Factor XIII, known as fibrin sealant, or "fibrin glue ' '.
  • fibrin glue is biocompatible, hemostatic, and does not interfere with normal healing, it suffers from poor mechanical strength. Synthetic cynaoacrylate adhesives have superior mechanical
  • bioadhesives include prolamine gels, which are based on
  • GRP Gelatin resorcinol formol
  • bioadhesive protein is first isolated from phenol glands of mussels of the genus Mytilus. The
  • isolated protein having a molecular weight of 120.000 to 140,000 daltons, is treated with collagenase, and then with trypsin, to yield, after gel filtration and dialysis, the decapeptide
  • a graft co-polymer comprising a poly(lysine), poly(allylamine),
  • chitosan, polydextran, or similar backbone is grafted with various peptides or the decapeptide disclosed by Waite '397, supra.
  • the protected decapeptides are obtained by
  • mussel adhesive precursor protein is cloned into a simple organism such as a bacteria, and the
  • the bacteria will usually then secrete the protein, which can be isolated and purified.
  • Pat. No. 5, 149,657 discloses construction of a DNA encoding a bioadhesive
  • the analog comprises three, ten, fifteen or twenty repeating
  • transformation a host inserted into a host (referred to as "transformation").
  • transformation the host is induced to
  • mussel M. edulis together with constructed expression vectors capable of expressing these precursor proteins in microorganisms, are cloned in microbial hosts. After isolation from the microbial broth, at least a portion of the tyrosine residues of the bioadhesive precursor proteins are then enzymatically hydroxylated by mushroom tyrosinase, mimicking the process
  • U.S. Patent No. 5,242,808 discloses an alternative scheme for
  • dsDNA double-stranded DNA encoding proteins having from about 50 to about 1500
  • the dsDNA is inserted into a vector, and the vector is used
  • the tyrosine residues of the produced protein are at least partially hydroxylated to dopamine by mushroom or Streptomyces antibioticus tyrosinase prior to cure.
  • bacterial or yeast cell such as
  • the protein may not be subjected to such post transitional modification and the protein may therefore not function as originally intended.
  • the protein may not be subjected to such post transitional modification and the protein may therefore not function as originally intended.
  • the protein may not be subjected to such post transitional modification and the protein may therefore not function as originally intended.
  • hydroxyproline likely contributes to the tertiary structure of the natural protein, as
  • proline in the produced proteins could conceivably be hydroxylated by chemical or enzymatic means to hydroxyproline, such transformation would add at least one
  • vectors comprising DNA sequences
  • bioadhesive protein precursor analog encoding the bioadhesive protein precursor analog are constructed and used to transform a microbial host, preferably E. coli. During growth, the host is contacted with a hypertonic growth media comprising hydroxyproline, resulting in assimilation and incorporation of
  • the bioadhesive precursor protein analog produced by this method comprises from
  • the bioadhesive precursor protein analog comprises from three to about one hundred, from three to about fifty, or from
  • Pro/Hyp is hydroxyproline. other protein analog, or mixtures thereof.
  • from about 1% to about 100%) from about 5% to about 75%, from about 15% to about 50%>, or from about 5%
  • hydroxyproline other proline analog, or a mixture thereof.
  • Hydroxyproline analogs of proline such as t ⁇ s-4-hydroxy-L-proline, ⁇ ' . -4-hydroxy-L-proline, tr ⁇ !>-3-hydroxy-L-
  • proline c/.y-3-hydroxy-L-proiine, and 3,4-dihydroxy-L-proline are of particular interest.
  • Other embodiments comprise vectors having the DNA sequences encoding the bioadhesive protein precursor analogs, E. coli hosts transformed with said vectors, the bioadhesive precursor protein analogs produced thereby, and adhesives formed from said bioadhesive precursor protein analogs.
  • FIG. 1 is a dsDNA sequence encoding a bioadhesive precursor protein decapeptide having the sequence Ala-Lys-Pro/Hyp-Ser-Tyr-Pro/Hyp-Pro/Hyp-Thr-Tyr-Lys.
  • FIG. 2 is a flow chart showing the assembly of a repetitive DNA sequence encoding a
  • bioadhesive precursor protein analog comprising decapeptides having the sequence Ala-Lys- Pro/Hyp-Ser-Tyr-Pro/Hyp-Pro/Hyp-Thr-Tyr-Lys.
  • FIG. 3 is a diagram of E. coli plasmid pLSM-6 containing a DNA sequence encoding 25 repeats of a bioadhesive precursor protein decapeptide having the sequence Ala-Lys-
  • FIG. 4 is a diagram of E. coli plasmid pLSM- 10 containing a DNA sequence
  • FIG. 5 is a flow chart showing the assembly of a repetitive DNA sequence encoding a
  • bioadhesive precursor protein analog comprising decapeptides having the sequence Ala-Lys-
  • FIG. 6 shows by SDS-PAGE analysis expression of protein precursor analogs comprising tr ⁇ S-4-L-hydroxyproline in pLSM-10 in E. coli.
  • FIG. 7 shows by SDS-PAGE analysis expression of protein precursor analogs comprising tr ⁇ /w-4-L-hydroxyproline (Hyp) and cw-4-hydroxy-L-proline (cis-4-Hyp) in
  • Vectors comprising synthetic dsDNA sequences encoding the bioadhesive precursor protein analog
  • microbial host preferably E. coli.
  • the transformed host is grown under conditions in which the protein is
  • precursor protein is then isolated, purified if necessary, and the tyrosine residues hydroxylated by chemical or enzymatic means prior to cure.
  • E. coli While E. coli is presently preferred as a host, both prokaryotic and eukaryotic cells
  • prokaryotic bacteria including but not limited to prokaryotic bacteria, eukaryotic cells such as
  • Saccharomyces cervisiae Saccharomyces carlsbergensis, and Schizosaccharomyces pombe, and additional eukaryotes such as insect cells including lepidopteran cell lines such as
  • baculovirus baculovirus
  • CHO cells COS cells, and NIH 3T3 cells.
  • the bioadhesive precursor protein analog produced by this method comprises from about 1% to about 50%. from about 5% to about 30%. or from about 10%) to about 20%) by total number of residues hydroxyproline, other proline analog, or mixtures thereof.
  • the bioadhesive precursor protein analog comprises a repeating decapeptide unit
  • bioadhesive precursor protein analog preferably comprises from about three to about 100,
  • bioadhesive precursor protein comprises repeat units of the above decapeptide interspersed with at least one
  • hexapeptide linker One particularly useful hexapeptide linker has the sequence
  • From one to about thirty, one to about fifteen, or one to about five repeat DNA units encoding for the decapeptide may be interspersed between the hexapeptide linker.
  • proline analog or a combination thereof.
  • proline such as tr ⁇ n. ⁇ -4-hydroxy-L-proline, c/.y-4-hydroxy-
  • analogs include, but are not limited to, cis- and tr ⁇ r ⁇ -4-fluoro-L-proline, cis- and trans-3- fluoro-L-proline, their D-analogs, 3,4-epoxyproline, and the like. Mixtures of two or more of any of the foregoing may also be used. For convenience, this specification uses the term
  • hydroxyproline to refer inclusively to tr ⁇ n.y-4-hydroxy-L-proline, cw-4-hydroxy-L-proline, tr ⁇ ra-3-hydroxy-L-proline, cw-3-hydroxy-L-proline, 3,4-dihydroxy-L-proline, their D-forms, and other proline analogs substituted with one or more hydroxy groups which are assimilated under hypertonic growth conditions and incorporated into proteins during translation.
  • Proline analogs refers inclusively to the above and other analogs, as long as
  • bioadhesive precursor protein analog is first produced by
  • the sequence is operably linked to a regulatory sequence that is
  • dsDNA is designed so as to encode for a precursor protein which is an analog of the naturally
  • precursor protein analog means a protein which may differ from the M. edulis protein in its exact amino acid sequence, but which comprises decapeptide repeating units which are common to the naturally occurring M.
  • the dsDNA sequence is further designed so as to incorporate preferred E. coli codons. A preferred sequence is shown in FIG. 1.
  • the dsDNA sequence encoding the bioadhesive precursor protein decapeptide is prepared by any of a number of known methods of DNA synthesis. A suitable method for the
  • synthesis of a dsDNA sequence is the methyl phosphite solid-phase method (Tetrahedron Letters, Vol. 21, pp. 719-722 (1980), and was used to synthesize the above-described dsDNA
  • the purified DNA is enzymatically phosphorylated at the 5'-end prior to subsequent ligation.
  • DNA sequences are then ligated to form multimer sequences having from three to about 100 repeats, from about three to about 50 decapeptide sequence repeats, from three
  • linker sequences providing unique restriction sites are added to the 5' and 3' ends to produce the sequence shown in FIG. 2. These sequences are then annealed, cleaved with the
  • expression vector i.e., pET-20b to generate pUSC-MAP
  • a plasmid that comprises ten of the
  • pLS - ⁇ contains the bioadhesive
  • FIG. 5 a hexapeptide having the sequence Lys-Tyr-Pro/ ⁇ yp-Ser-Ala-Lys-C0 2 H is shown in FIG. 5.
  • Synthesis of DNA encoding for the decapeptide sequence -Ala-Lys- Pro Hyp-Ser-Tyr-Pro Hyp-Pro Hyp-Thr-Tyr-Lys-CO 2 H is followed by ligation into multimers having from one to about thirty, one to about fifteen, or one to about five DNA
  • Linkers encoding for restriction sites e.g.,
  • restriction site for Dde I is ligated immediately adjacent each end of
  • restriction enzyme Dde I results in DNA which can then be self-ligated using the cut ends.
  • Self-ligation results in DNA sequences having from one to about thirty, one to about fifteen,
  • DNA units encoding for bioadhesive protein precursor DNA units encoding for bioadhesive protein precursor.
  • Other DNA/amino acid sequence/restriction enzyme combinations as embodied by the above-described hexapeptide
  • sequence/Dde 1 combination may be used as long as the resultant amino acid sequence does not inhibit protein expression, isolation, adhesiveness, or other desired bioadhesive precursor
  • Amino acid sequences may be up to fifty, and even greater number of
  • the expression vector containing the inserted multimers coding for the bioadhesive precursor protein analog and optional hexapeptide is then used to transform E. coli cells by
  • the transformed E. coli cells are cultured under
  • RNA messenger RNA
  • ribosomes made of ribosomal RNA (rRNA). Transfer RNA (tRNA) meanwhile links to free amino acids in the cell pool to form amino acid/tRNA complexes.
  • rRNA ribosomal RNA
  • tRNA Transfer RNA
  • tRNA mediated by tRNA.
  • Each of up to about twenty amino acids is distinguished from other amino acids and charged to tRNA by enzymes known as aminoacyl-tRNA synthetases.
  • Amino acid/tRNA complexes are generally quite specific, such that normally only a molecule
  • a cell capable of producing the desired protein when contacted with a preferably hypertonic growth
  • hydroxylation of proline not present in the native production system of a recombinant host such as E. coli, Saccharomyces cervisiae, and eukaryotic cell lines including Spodoptera frugiperda, Tricholplasia, ni, Heliothis virescens, , and others.
  • a culture of the transformed E. coli strain is grown in a rich
  • proline analog content may be adjusted and optimized by the nature and concentration of proline analogs added at
  • the total hydroxyproline content of the bioadhesive precursor protein may be adjusted by the addition of suitable mixtures of proline and
  • analogs may result in a protein having incorporated therein a mixture of the protein analogs.
  • proline analogs suitable for producing such proteins is empirically determined, and will depend on the relative rates of analog uptake, aminoacyl t-RNA charging, and
  • Novel proteins are available using this method, in that the naturally-occurring bioadhesive proteins are known to comprise a mixture
  • residues consist of proline and from 1% up to 100% tr* y-4-hydroxy-L-proline but no other
  • proline analogs Use of a treatment mixture comprising exclusively 3-hydroxy-L-proline (with or without proline) yields a bioadhesive precursor protein wherein the Pro/Hyp residues consist of proline and from 1% up to 100% 3-hydroxy-L-proline and no other proline analogs.
  • the protein is preferably
  • the recovered protein is preferably subjected to cyanogen bromide cleavage to remove extraneous peptide sequences.
  • bioadhesive precursor protein analog is converted to adhesive by methods known in the art, for example by chemical or enzymatic hydroxylation of tyrosine residues and
  • Enzymatic hydroxylation of tyrosine may be effected in the presence of mushroom tyrosinase as described by Ito et al. in
  • first (left-most) amino acid is the N-terminal amino acid and the last (right-most) amino acid is the C -terminal amino acid.
  • a DNA sequence was synthesized using codon preferences favorable for expression
  • MAP analog Ala-Lys-Pro/Hyp-Ser-Tyr-Pro/Hyp-Pro/Hyp-Thr-Tyr-Lys
  • DNA oligomer (800 pmol) was 5'-phosphorylated in a 25 ⁇ L reaction mixture containing T4
  • Proline Plasmid pLSM-10 obtained in Example 1 (FIG. 4) was used to transform proline
  • auxotrophic E. coli strain JM109F-(pro _ ) A 20 mL culture of the transformed E. coli strain was grown in LB medium until an OD 600 of 0.75 - 1.00 was reached. The cells were
  • M9 minimal medium M9 salts, 2% glucose, 0.01
  • thiamine supplemented with the 20 natural amino acids minus proline at 50 ⁇ g/mL
  • the medium replaced by an equal amount of the identical M9 minimal medium with the additon of 400 mM NaCl.
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • tr ⁇ n. ⁇ -4-hydiOxy-L-proline was added to provide a final concentration of 15 mM.
  • reverse-phase e.g., C-4
  • HPLC high pressure liquid chromato graphy
  • Proline The protein of Example 3 is concentrated to 0.8 mg/mL in 0.1 M phosphate buffer
  • Example 3 or the stored dopamine-containing protein after tyrosinase incubation is effected
  • FIG. 3 was used to transform proline auxotrophic E. coli strain JM109(DE3)pLysS (pro-).
  • a 20 mL culture of the transformed E. coli strain was grown in LB medium until an OD 600 of about 1.0 was reached. The cells were harvested by centrifugation, washed with M9 minimal
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • lysis buffer 50 mM sodium phosphate buffer. pH 6.4. 10 mM EDTA, 100 mM NaC l , 5 mM 2-mercaptoethanol, and 250 ⁇ g/mL lysozyme. Soluble and insoluble cell fractions from one experiment are shown in
  • Protein in the insoluble cell fraction is solubilized by addition of 0.8 M acetic acid, 2 M urea, and
  • bioadhesive precursor protein I further purified by standard chromatographic methods
  • Example 6 Purified as in Example 6 is concentrated to 0.8 mg/mL in 0.1 M phosphate buffer and
  • the protein may also be stored in 5% acetic acid or in borate buffer adjusted to pH 8.5.
  • Complete cure i.e, cross-linking of the dopamine to produce a cross-linked gel or to attach a
  • FIG. 3 was used to transform proline auxotrophic E. coli strain JM109(DE3)pLysS (pro-).
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • proline to a concentration of 15 mM, and proline to a concentration of 10 ⁇ M.
  • the cells were incubated for an additional 4 hours at 37°C with vigorous shaking (250 ⁇ m). The cells
  • ⁇ were harvested by centrifugation, and lysed by sonication in lysis buffer (50 mM sodium
  • the cell pellet from Example 8 is resuspended in one-fiftieth of the volume of the original culture in lysis buffer (50 mM sodium phosphate, pH 6.5, 100 mM NaCl , 10 mM
  • Protein in the insoluble cell fraction is solubilized by addition of 0.8 M acetic acid, 2 M urea, and dialyzed first against 0.5 M acetic acid (2 changes, 3 hours each, 4 °C) and lyophilized to
  • the dry protein material is redissolved in a suitable volume of 0.1 M HC1, and a 5
  • bioadhesive precursor protein I further purified by standard chromatographic methods
  • Example 9 is concentrated to 0.8 mg/mL in 0.1 M phosphate buffer and
  • the protein may also be stored in 5% acetic acid or in borate buffer adjusted to pH 8.5.
  • Complete cure i.e, cross-linking of the dopamine to produce a cross-linked gel or to attach a

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Abstract

L'invention concerne une technique qui permet de produire des analogues de protéines précurseurs bioadhésives renfermant de l'hydroxyproline. La technique consiste à construire des vecteurs renfermant des séquences d'ADN codant pour lesdits analogues et à les utiliser pour transformer un hôte microbien, de préférence E. Coli. Durant la croissance, l'hôte est mis en contact avec un milieu hypertonique renfermant de l'hydroxyproline, ce qui entraîne l'assimilation de cette dernière par la protéine précurseur bioadhésive et son incorporation dans ladite protéine durant la traduction. L'incorporation d'hydroxyproline dans les protéines précurseurs bioadhésives durant leur production est avantageuse en ce que la modification post-traductionnelle des protéines n'est pas nécessaire.
PCT/US1999/020463 1998-09-09 1999-09-07 Analogues recombinants de proteines bioadhesives contenant de l'hydroxyproline WO2000015789A1 (fr)

Priority Applications (1)

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AU59100/99A AU5910099A (en) 1998-09-09 1999-09-07 Recombinant bioadhesive protein analogs comprising hydroxyproline

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US09/149,973 1998-09-09
US14997398A 1998-09-11 1998-09-11

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1727830A1 (fr) * 2004-03-26 2006-12-06 Postech Foundation Bioadhesif de moule
CN106916564A (zh) * 2017-03-15 2017-07-04 吉林大学 一种基于氧化型谷胱甘肽与无机多金属氧簇的复合仿生黏合剂及其制备方法
WO2021110061A1 (fr) * 2019-12-02 2021-06-10 Jiangyin Usun Pharmaceutical Co., Ltd. Peptides et leur utilisation dans le traitement d'une inflammation

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988003953A1 (fr) * 1986-11-24 1988-06-02 Genex Corporation Bioadhesifs
WO1988007076A1 (fr) * 1987-03-12 1988-09-22 Genex Corporation Production d'analogues de proteines precurseur bioadhesifs par des organismes mis au point genetiquement
US5410023A (en) * 1989-06-27 1995-04-25 Burzio; Luis O. Peptide useful as adhesive, and process for the preparation thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988003953A1 (fr) * 1986-11-24 1988-06-02 Genex Corporation Bioadhesifs
WO1988007076A1 (fr) * 1987-03-12 1988-09-22 Genex Corporation Production d'analogues de proteines precurseur bioadhesifs par des organismes mis au point genetiquement
US5410023A (en) * 1989-06-27 1995-04-25 Burzio; Luis O. Peptide useful as adhesive, and process for the preparation thereof

Non-Patent Citations (2)

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Title
INOUE K ET AL: "Mussel adhesive plaque protein gene is a novel member of epidermal growth factor-like gene family.", J BIOL CHEM, MAR 24 1995, 270 (12) P6698-701, UNITED STATES, XP002127537 *
WAITE J H: "EVIDENCE FOR A REPEATING 3,4-DIHYDROXYPHENYLALANINE-AND HYDROXYPROLINE-CONTAINING DECAPEPTIDE IN THE ADHESIVE PROTEIN OF THE MUSSEL, MYTILUS EDULIS L", JOURNAL OF BIOLOGICAL CHEMISTRY. (MICROFILMS),US,AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS, BALTIMORE, MD, vol. 258, no. 5, pages 2911-2915, XP000651279 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1727830A1 (fr) * 2004-03-26 2006-12-06 Postech Foundation Bioadhesif de moule
EP1727830A4 (fr) * 2004-03-26 2007-08-22 Postech Foundation Bioadhesif de moule
US7622550B2 (en) 2004-03-26 2009-11-24 Postech Foundation Mussel bioadhesive
CN106916564A (zh) * 2017-03-15 2017-07-04 吉林大学 一种基于氧化型谷胱甘肽与无机多金属氧簇的复合仿生黏合剂及其制备方法
WO2021110061A1 (fr) * 2019-12-02 2021-06-10 Jiangyin Usun Pharmaceutical Co., Ltd. Peptides et leur utilisation dans le traitement d'une inflammation

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