WO1988007076A1 - Production d'analogues de proteines precurseur bioadhesifs par des organismes mis au point genetiquement - Google Patents

Production d'analogues de proteines precurseur bioadhesifs par des organismes mis au point genetiquement Download PDF

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Publication number
WO1988007076A1
WO1988007076A1 PCT/US1988/000876 US8800876W WO8807076A1 WO 1988007076 A1 WO1988007076 A1 WO 1988007076A1 US 8800876 W US8800876 W US 8800876W WO 8807076 A1 WO8807076 A1 WO 8807076A1
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analog
bioadhesive
protein
precursor protein
bioadhesive precursor
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PCT/US1988/000876
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English (en)
Inventor
Kathy J. Maugh
David M. Anderson
Susan L. Strausberg
Robert Strausberg
Tena Wei
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Genex Corporation
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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
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces

Definitions

  • This invention relates to the production of bio ⁇ adhesives that can be employed to bond substances in wet environments.
  • the bioadhesives of the inven ⁇ tion are employed as marine adhesives, biomedical adhesives or dental adhesives.
  • the invention further relates to the microbial production of bioadhesive precursor proteins that can be converted to bioadhesives by chemical or enzymatic treatment.
  • adhesive should be cured and it should maintain both its adhesivity and cohesivity under the conditions of use. Curing is the altering of the physical properties of an adhesive by chemical or enzymatic means. In the case of the bioadhesives produced by the procedures described herein, curing is likely to be due to the cross-linking of adjacent uncured adhesive molecules by catalytic and/or chemical agents. Curing may also involve adhesive cross-linking with the substrate.
  • Marine mussels and other sessile invertebrates have the ability to secrete adhesive substances by which they affix themselves to underwater objects.
  • mussels of the genus Mvtilus e.g., the species Mvtilus edulis and Mytilus californianus. deposit an adhesive substance from the mussel foot that becomes cured, forming a permanent attachment to the substrate.
  • a major component of the adhesive deposited by M. edulis has been identified as a hydroxylated protein of about 130,000 daltons (Waite, J. H., J. Biol. Chem., 258:2911- 2915 (1983)).
  • Bioche ical analysis of the M. edulis bioadhesive protein has shown it to be rich in lysine (20 residues/100) and hydroxylated a ino acids (60 residues/100) (Waite, J. H. , supra) . At least a portion of the hydroxylated residues are 3,4-dihydroxyphenyl- alanine (DOPA) and hydroxyproline, formed by post-trans- lational hydroxylation of tyrosine and proline residues, respectively. It is believed that post-translational hydroxylation, particularly of the tyrosine residues, is important in defining the adhesive properties of the protein (Waite, J.
  • DOPA 3,4-dihydroxyphenyl- alanine
  • hydroxyproline formed by post-trans- lational hydroxylation of tyrosine and proline residues, respectively. It is believed that post-translational hydroxylation, particularly of the tyrosine residues, is important
  • U.S. Patent No. 4,585,585 describes a procedure for preparing a bioadhesive polymer by chemically linking decapeptide units produced by the enzymatic digestion of isolated mussel adhesive protein.
  • a bioadhesive protein is first isolated from phenol glands of mussels of the genus Mytilus using the protein purification procedures described by Waite and Tanzer in Science. 212:1038 (1981) .
  • the isolated bioadhesive having a molecular weight of 120,000 to 140,000 daltons, is first treated with collagenase, which reduces its molecular weight by about 10,000 , daltons.
  • the treated protein is then digested with trypsin, and the digested protein sub ⁇ jected to gel filtration dialysis to isolate decapep- tides of the general formula
  • decapeptides produced in this manner are then poly ⁇ merized by the use of chemical linking groups such as glutaraldehyde, oligopeptides, amino acids or other bifunctional linking groups to produce bioadhesives containing up to about 1,000 such decapeptide units.
  • chemical linking groups such as glutaraldehyde, oligopeptides, amino acids or other bifunctional linking groups to produce bioadhesives containing up to about 1,000 such decapeptide units.
  • This invention involves the production, using techniques of recombinant DNA technology, of bioadhesive precursor proteins which are analogous to the non- hydroxylated polyphenolic adhesive protein produced by marine invertebrates.
  • the invention includes the DNA sequences encoding the bioadhesive precursor protein analogs, the vectors comprising said sequences, hosts transformed with said vectors, the bioadhesive precursor proteins, the hydroxylated bioadhesive proteins and methods of producing the precursor protein, methods of producing the hydroxylated protein, methods of producing adhesives, and methods of using the adhesives.
  • the bioadhesive precursor protein which is produced by the process of the invention comprises a sequence of from about 50 to about 1500 amino acid residues which comprise from about 20% to 40% proline residues; from about 10% to 40% lysine residues; from about 10% to 40% tyrosine residues; and from 0 to about 40% amino acid residues other than proline, lysine and tyrosine.
  • the bioadhesive precursor protein of the invention is comprised of repeating polypeptide sequences. Each polypeptide contains about 20-40% proline residues, 10-40% lysine residues, and 10-40% tyrosine residues and 0-40% of other amino acid residues.
  • interspersed throughout are polypeptide linking groups.
  • the protein produced by the process of the inven ⁇ tion can be employed as a bioadhesive precursor.
  • the adhesive properties of the protein are enhanced by hydroxylating at least a portion of the tyrosine residues to 3,4-dihydroxyphenylalanine (DOPA) and, optionally, at least a part of the proline residues to 3- or 4-hydroxyproline by chemical or enzymatic means.
  • DOPA 3,4-dihydroxyphenylalanine
  • the hydroxylated protein is cured to produce the desired physical properties in the bioadhesive.
  • the hydroxylated bioadhesive precursor protein is analogous to the adhesive protein isolated from the phenol gland of the mussel M. edulis (Waite, J. H., J. Biol. Chem.. 258:2911-2915 (1983).
  • the bioadhesive precursor protein analog is produced by the insertion into an appropriate host, suc ⁇ r as E. coli, £. cerevisiae. £. subtilis. A. nicer. P. pastorus or mammalian cells of a replicable expression vector containing a chemically synthesized double- stranded DNA (dsDNA) sequence coding for the desired protein and expression of the synthetic dsDNA sequence in the host to yield the protein.
  • the synthetic dsDNA sequence encoding the bioadhesive precursor protein of the invention may be constructed of codons which are selected to optimize expression and provide for stable reproduction of the genetic information in the par ⁇ ticular host employed.
  • the dsDNA sequence encoding the bioadhesive precur ⁇ sor protein can be linked, at its 5' end, to a sequence which encodes an N-terminal portion of a host protein in order to facilitate transcription initiated at a host promoter*
  • the expressed protein will constitute a fusion of the bioadhesive precursor protein and the host protein fragment.
  • the host protein frag ⁇ ment may include a signal peptide which, in the case of a host such as S. cerevisiae, facilitates secretion of the expression product across the cell membrane and into the surrounding medium, with attendant cleavage of the signal peptide.
  • Insertion, between the sequences encod ⁇ ing the bioadhesive precursor protein and the host protein fragment, of a dsDNA sequence encoding an amino acid sequence which is specifically cleavable by chemical or enzymatic methods provides a means of cleav ⁇ age to separate the bioadhesive precursor protein from the host protein fragment.
  • Figure 1 is a diagram of E ⁇ . coli plasmid pGX2287, containing the OL/PR promoter and encoding a trpB- chymosin fusion protein.
  • Figure 2 is a flow chart describing the assembly of a repetitive DNA sequence encoding a bioadhesive precur ⁇ sor protein analog.
  • Figure 3 is a diagram of plasmid YpGX265GAL4, containing a hybrid GALl/MF-alphal promoter, a PH05 signal coding sequence, a GAPDH transcription termin ⁇ ator, and selectable markers and replication origins for S. cerevisiae and E_j. coli.
  • Figure 4 is a diagram of E___ coli plasmid pGX2213, containing the trpB and trpA regions of the tryptophan synthetase operon and the tac promoter.
  • Figure 5 is a diagram of S_ s . cerevisiae-E. coli shuttle vector YpGXl.
  • Figure 6 is a diagram of S cerevisiae plasmid YpGX ⁇ O containing a portion of the phosphoglycerate kinase gene.
  • Figure 7 depicts a method of inserting synthetic DNA sequences encoding bioadhesive precursor protein analogs into IL. coli plasmid pGX2287 to generate coding sequences for a tribrid fusion protein.
  • Figure 8 represents the DNA sequence and amino acid sequence of the portion of the tribrid gene of pGX2J54 which codes for a 10 repeat decapeptide.
  • Figure 9 represents the amino acid sequence of the bioadhesive precursor protein analog encoded by pGX2365, after cyanogen bromide cleavage.
  • Figure 10 represents a Western blot analysis for several bioadhesive precursor protein analogs produced in E_i. coli.
  • Figure 11 is a diagram of an Ml3-based vector, MGX436, containing the S_j_ cerevisiae MF-alphal promoter.
  • Figure 12 represents SDS-polyacrylamide gel analysis for several bioadhesive precursor protein analogs produced in S ⁇ . cerevisiae.
  • Figure 13 represents the DNA sequence and trans ⁇ lated amino acid sequence for a gene identified as cDNA clone 14-1 which codes for a bioadhesive precursor protein of M ⁇ . edulis.
  • Figure 14 represents the DNA sequence and trans ⁇ lated amino acid sequence for a gene identified as cDNA clone 52, which codes for a bioadhesive precursor pro ⁇ tein of I edulis.
  • Figure 15 represents the DNA sequence and trans ⁇ lated amino acid sequence for a gene identified as cDNA clone 55, which codes for a bioadhesive precursor prot ⁇ ein of Mj. edulis.
  • Figure 16 represents the DNA sequence and trans ⁇ lated amino acid sequence for a gene identified as cDNA clone 56, which codes for a bioadhesive precursor pro ⁇ tein of U s . edulis.
  • Figure 17 represents the DNA sequence of the bio ⁇ adhesive precursor protein analog gene of plasmid PGX2385 of Example 7, and the amino acid sequence of the bioadhesive precursor protein analog encoded for there ⁇ by.
  • Figure 18 represents the DNA sequence of the bio ⁇ adhesive precursor protein analog gene of plasmid pGX2386 of Example 7, and the amino acid sequence of the bioadhesive precursor protein analog encoded for thereby.
  • Figure 19 represents the DNA sequence of the bio ⁇ adhesive precursor protein analog gene of plasmid pGX2393 of Example 7, and the amino acid sequence of the bioadhesive precursor protein analog encoded for thereby.
  • the bioadhesive precursor protein produced by the process of the invention comprises a sequence of from about 50 to about 1500 amino acid residues, preferably from about 600 to about 900 amino acid residues, arranged in repeating polypeptides, preferably repeating decapeptides and hexapeptides.
  • the protein, and prefer ⁇ ably each polypeptide is comprised of about 20% to about 40% proline residues.
  • proline residues impart flexibil ⁇ ity to the bioadhesive and render the molecule non- globular, so that the bioadhesive is capable of conform- ing to the surface of a substrate and interacting with other adhesive molecules.
  • the protein and preferably each polypeptide is also comprised of about 10% to about 40% lysine residues.
  • the lysine residues render the bioadhesive basic, which assists in bonding to under ⁇ water surfaces, which are generally coated with a thin film of acidic biological material. It also provides reactive groups through which the protein can be cross- linked w during the curing process.
  • the protein and preferably each polypeptide also is comprised of about 10% to • about 40% tyrosine residues.
  • the phenolic tyrosine residues provide hydrogen bonding capability to the bioadhesive.
  • both the proline and tyrosine residues provide sites for hydroxylation. The addition of a hydroxyl group on the tyrosine to form DOPA is believed to enable the bioadhesive to strongly displace water molecules from the surface of a sub ⁇ strate.
  • the protein and preferably each polypeptide comprises from about 0 to about 40% other amino acid residues.
  • these residues if present, have non-reactive aliphatic side chains, e.g., alanine, and hydroxyl-containing amino acids, e.g., serine and threonine. These residues are preferably distributed throughout the protein chain so that no more than about four occur together in any given polypeptide sequence.
  • Non-preferred amino acids are acidic amino acids, i.e., aspartic acid and gluta ic acid, and cysteine.
  • the bioadhesive precursor protein analog is pro ⁇ substituted by inserting a synthetic dsDNA sequence encoding the protein into a replicable expression vector in which it is operably linked to a regulatory sequence that is capable of directing expression of the encoded protein in appropriate host cells.
  • host cells for example, £. coli or S_. cerevisiae. can then be trans ⁇ formed with the expression vector, grown up and sub ⁇ jected to conditions under which the protein is expressed.
  • the term "recombinant protein” is intended to mean a protein produced by a host transformed with a recombinant repli ⁇ cable expression vector.
  • the dsDNA sequence encoding the bioadhesive precur ⁇ sor protein may be prepared by any of the known methods of DNA synthesis.
  • a suitable method for synthesizing the dsDNA sequence is the phosphite solid-phase method (Tetrahedron Letters, 21:719-722 (1980)).
  • the dsDNA is characterized by the fact that it codes for a protein which is an analog of a naturally occurring adhesive protein which has a repeating structure.
  • analog is intended a protein which differs from the naturally occurring protein in its exact amino acid sequence but which includes polypeptide repeating units which are common to a non-posttranslationally modified naturally occurring adhesive protein which has a repeat ⁇ ing structure.
  • bioadhesive precursor protein analog proteins produced in genetically engineered organisms which comprise repeat ⁇ ing polypeptide units which are identical or similar to those found in naturally occurring protein adhesives prior to posttranslational hydroxylation.
  • Particular polypeptide analogs, and the DNA sequences coding for these analogs, are presented in Figures 8, 17, 18, and 19.
  • bioadhesive precursor protein analogs of the M_ s _ edulis protein includes analogs of any and all natural protein adhesives which have a repeating poly ⁇ peptide structure.
  • the techniques described herein are useful in the development of analog precursors and the hydroxylated derivatives thereof for other protein adhesives from marine invertebrates.
  • the invention further includes bioadhesive pre ⁇ cursor protein analog derivatives as well.
  • bioadhesive precursor protein analog derivative(s) is intended those polypeptides which differ from the bioad ⁇ hesive precursor protein analogs by one or more amino acids but which still retain the basic properties of same.
  • This approach was used to assemble synthetic DNA sequences encoding the polypeptide (ala-lys-pro-ser-tyr- pro-pro-thr ⁇ tyr-lys) N where N indicates the number of direct repeats of this decapeptide sequence.
  • This decapeptide is a component of the polyphenolic adhesive protein of M.. edulis and was identified in tryptic digests of the natural protein (U.S. Patent No. 4,585,585). This approach could also be used to assemble synthetic DNA sequences encoding repeats of other polypeptides.
  • the inventors anticipated that a cloned multimer coding sequence containing for example, 20 repeats of a 10-codon sequence might be unstable in £. coli.
  • a cloned multimer coding sequence containing for example, 20 repeats of a 10-codon sequence might be unstable in £. coli.
  • five different oligonucleotides were synthesized, using different codon combinations. How ⁇ ever, use of one particular oligonucleotide (GCG AAA CCA AGT TAC CCA CCG ACC TAC AAA) encoding the decapeptide resulted in the most efficient assembly of the multimer coding sequence, and the resulting repetitive DNA sequence was found to be stable in E. coli.
  • DNA sequencing and/or restriction enzyme analysis of clones obtained by this approach indicated that DNA fragments encoding up to nine decapeptide repeats had been cloned in jjjJ. coli and up to three decapeptide repeats had been cloned in S. cerevisiae. However, many of the clones had errors in the DNA sequences, causing incorrect codons, frame shifts or termination codons. Therefore, an improved approach for generating a homo ⁇ geneous 20-decapeptide repeat coding sequence was developed.
  • the second approach, represented in Example 4 was to:
  • the synthetic dsDNA encoding the bioadhesive pre ⁇ cursor protein analog is inserted into either the E. coli or S_. cerevisiae expression vectors under the control of a regulatory sequence containing a promoter, ribosome binding site and translation initiation signal capable of effecting expression in the selected host.
  • the expression vector can be selected from plasmids and phages, with plasmids generally being preferred.
  • the synthetic dsDNA encoding the protein may be linked, at its 5' end, to a sequence encoding an N-terminal portion of the microbial protein which is normally under the control of the particular regulatory sequence employed.
  • the 5 « end preceding the sequence encoding the bioadhesive precursor protein analog may also encode a signal peptide for a normally secreted protein which should allow the bioadhesive protein precursor analog to be secreted.
  • the genes contain a 5' segment of the highly expressed trpB gene to promote efficient translation initiation, followed by the synthetic bio ⁇ adhesive precursor protein analog gene, and a 3' region encoding the 159 carboxy terminal amino acids of bovine chymosin. Methionine codons are located on either end of the bioadhesive precursor protein segment so that cyanogen bromide cleavage can be used to release the bioadhesive precursor protein analog from the tribrid fusion protein. Additionally, the plasmid contains a synthetic trpt sequence 3' of the gene to stabilize the mRNA and the trpED genes that effectively stabilize the plasmids in the GX3015 deltatrpED102 host when media without tryptophan is utilized.
  • the promoter is a hybrid lambda OL/PR promoter (fully described in co- pending, commonly assigned U.S. Patent Application No. 534,982) that is regulated by the temperature-sensitive cI857 repressor produced by a defective lambda lysogen in the GX3015 host.
  • Other specific vector constructions for the expression of the bioadhesive precursor protein analogs will be apparent to those skilled in the art based on the description herein. As a general rule, however, it is advantageous to construct the vector by inserting the bioadhesive precursor protein coding region as an in-frame fusion with another gene that is under the control of an efficient promoter.
  • the fusion is constructed such that the encoded fusion protein contains a methionine residue at the 5' end of the bioadhesive precursor protein segment.
  • the recovered bioadhesive precursor protein can thus be treated with cyanogen bromide, using conditions well- known in the art, to remove extraneous amino acid sequences.
  • cyanogen bromide cleaves proteins at methionine residues. Since there are no internal methionine residues within the bioadhesive precursor protein itself, this protein remains intact.
  • oligonucleotides were synthesized that encode the deca ⁇ peptide sequence and 5• and 3• linker sequences that provide unique restriction sites. These oligonucleo ⁇ tides were annealed and ligated with an 1. coli expres ⁇ sion vector to generate pGX2346, a plasmid that contains three decapeptide coding repeats. The 5' linker encodes a Notl site and the 3' linker encodes a Nael site.
  • Notl and Nael sites were chosen for the linkers because they are unique sites in the plasmids and therefore, simplify the ligations to increase the synthetic gene length.
  • a plasmid with a five decapeptide repeat gene (pGX2348) was doubled to a ten repeat gene (pGX2354) with the thr-pro-ala linker between two five decapeptide repeat genes by simply ligating Notl/DNA polymerase I treated pGX2348 DNA with another aliquot of pGX2348 DNA digested with fiael, followed by digestion with Pvul (a site in the bla gene of pGX2348) and ligation again at low DNA concentration to favor recircularization of the plasmid.
  • the decapeptide observed by Waite is encoded in this sequence, but many other decapeptides are also encoded.
  • This heterogeneity in the natural protein sequence indicates that a family of bioadhesive precursor protein analogs could be produced that are related to the natural polyphenolic protein but differ in the frequency of certain decapeptides and hexapeptides and the mole ⁇ cular weight of the protein. This may allow novel adhesive proteins to be specifically designed for a given application. Therefore, in another embodiment, the general approach described above was used to assemble synthetic genes encoding bioadhesive precursor protein analogs composed of several different decapep ⁇ tide and hexapeptide sequences and of various molecular weights (see Example 7) .
  • the expres ⁇ sion vector can carry the entire coding region for the bioadhesive precursor protein analog o - a coding region for a fragment thereof.
  • the fragment con ⁇ tains at least enough of the coding sequence to code for 100 amino acids corresponding to the bioadhesive precur ⁇ sor protein analog.
  • the expression vector containing the inserted dsDNA coding for the bioadhesive precursor protein is used to transform a host by known techniques of transformation.
  • the expression vector provided by the invention is used to transform any suitable host microorganism, using known means, to produce a transformant.
  • Suitable host organisms include, for example, £. coli or other related gram-negative organisms such as Salmonella. Klebsiella. Erwinia. etc.
  • the assembled DNA sequence encoding the bioadhesive precursor protein analog may be inserted into an expression vector that functions in a yeast such as Saccharomyces cerevisiae.
  • a yeast such as Saccharomyces cerevisiae.
  • Typical vectors of this type are disclosed in co-pend ⁇ ing, commonly assigned U.S. application Serial No. 918,147, filed on October 14, 1986, and having the title "Composite Yeast Vectors,” incorporated by reference herein.
  • One preferred vector for expression of bio ⁇ adhesive precursor protein analogs in yeast comprises the yeast shuttle vector YpGX265GAL4 (ATCC #67233) ( Figure 3). This vector is characterized by a promoter that is a hybrid derived from the S.
  • the regulatory gene comprises the GAL4 gene which encodes the GAL4 protein, a positive regulator of the GAL1-MF- alphal hybrid promoter.
  • the terminator in the YpGX265GAL4 vector system is derived from synthetic DNA and is based on the S. cerevisiae GAPDH transcription terminator.
  • the signal encoding sequence, also derived from synthetic DNA, is based on the S_. cerevisiae PH05 signal. Codons are designed substantially for usage preference in S. cerevisiae.
  • the YpGX265GAL4 vector contains the LEU2 gene, a marker for plasmid selection in S. cerevisiae. It also contains DNA derived from S. cerevisiae 2-micron plasmid which provides a plasmid replication origin for S_. cerevisiae.
  • the vector is further characterized by the E. coli replication origin derived from pJBD207, and an £• coli selectable marker which is ampicillin resis ⁇ tance, also derived from pJBD207.
  • yeast expres ⁇ sion module including the GALl/MF-alphal hybrid promoter and PH05 signal encoding sequence are removed from vector YpGX265GAL4 as a single restriction fragment by digestion with restriction endonucleases Hindlll and BamHI and this fragment is ligated with M13mp9 which has also been digested with Hindlll and BamHI.
  • the bioad> hesive precursor analog protein coding sequence is removed from an iL.
  • coli expression vector such as pGX2365 by restriction endonuclease digestion and is positioned in the M13 vector carrying the yeast expres ⁇ sion module so that an in-frame fusion is generated between the yeast signal and bioadhesive precursor protein analog coding sequences.
  • Generation of the desired fusion coding sequence may involve the use of oligonucleotide linkers and/or oligonucleotide-directed mutagenesis.
  • the yeast expression module-fusion protein coding sequence is then excised from the M13 based vector and inserted in the yeast expression vector YpGX265 so that the yeast glyceraldehyde-3-phosphate dehydrogenase transcription terminator is situated downstream from the end of the fusion protein coding region.
  • This vector includes replication origins and selectable markers for plasmid maintenance for both yeast and E_s. coli. in a final step the yeast GAL4 gene is added to the expression vector as a Hindlll restric ⁇ tion fragment.
  • the expression vector may comprise the entire coding region for the bioadhesive precursor protein analog or coding regions for fragments thereof.
  • Saccharomvces strains carrying mutations in the LEU2 structural gene may be transformed with this plasmid, utilizing standard methods.
  • the resulting yeast strain may be grown in an appropriate medium (YNBD, containing 0.7% yeast nitrogen base, 2% glucose, and appropriate nutritional supple ⁇ ments) to maintain the plasmid.
  • the transformed yeast strain may be grown in an appropriate medium.
  • One suitable medium contains 1% yeast extract, 2% peptone, 1% glucose, and 1% galactose.
  • the transformant microorganism (E. coli or yeast) is cultured under conditions suitable for growth and expression of the bioadhesive precursor protein analog gene. After the protein has been expressed, it is recovered from the transformant cells by known methods such as mechanical or chemical lysis of the cells.
  • the protein can be purified using procedures known in the art, including well-known chromatographic procedures.
  • the bioadhesive precursor protein analog is preferably purified to homogeneity or near homogeneity. In the case of a fusion protein, the recovered protein can be subjected to cyanogen bromide cleavage to remove extraneous peptide sequences.
  • the recovered bioadhesive precursor protein analog is converted to a bioadhesive by hydroxylation.
  • hydroxylation converts tyrosine residues to DOPA residues and, option ⁇ ally a portion of the proline residues to hydroxyproline residues.
  • DOPA hydroxyl groups are believed to displace water at the bond surfaces, thus contributing to the excellent wet strength of the adhesive, and DOPA residues oxidized to quinones participate in inter- molecular cross-linking which cures the adhesive and imparts cohesivity.
  • Any suitable chemical or enzymatic means for effecting hydroxylation can be employed. It is prefer ⁇ red, however, to effect hydroxylation enzymatically using an enzyme such as mushroom tyrosinase or Strep- tomyces antibioticus tyrosinase. Enzymatic hydroxyla ⁇ tion procedures using these enzymes are carried out as generally described by Ito et al.. Biochem. J. 222:407- 411 (1984) and Marumo and Waite, Biochem. Biophys. Acta 852:98-103 (1986). Preferably, at least about 10% of the tyrosine residues are hydroxylated.
  • the mushroom tyrosinase can be removed from the protein using known procedures such as binding to a LH-Sephadex 60 column followed by elution with 0.2 M acetic acid or by mem ⁇ brane filtration. -— ⁇ *
  • the invention is intended to include analogs of any and all bioadhesive precursor proteins which have a repeating structure.
  • the skilled routineer can now deduce an appropriate DNA sequence, synthesize oligonucleotides encoding that sequence, assemble a synthetic gene coding for repeating polypeptide sequences, and express the repeating sequences.
  • the present invention thus makes available microbial production of bioadhesive precursor protein analogs of any and all natural protein adhesives which have a repeating structure.
  • bioadhesives produced by the methods of this invention can be used in a conventional manner and, if desired, may be admixed with conventional synthetic polymer adhesives, fillers, coacervates and/or adjuvants generally employed in adhesives. They are particularly useful where performance in wet environments is desired, such as marine adhesives or adhesives for medical or dental use, or protective coatings.
  • the bioadhesive protein can be lyophilized for reconstitution as an adhesive formulation at a later date. It can be employed as an . adhesive, a sealant, or as an adhesive primer in the form of a solution in a suitable solvent with or without other adhesive sub ⁇ stances.
  • suitable solvents for the bioadhesive include water or aqueous solutions of alcohols such as methanol, ethanol, propanol, and the like, acetone, DMSO, dimethyl formamide, and the like.
  • the bio ⁇ adhesive protein is present in the solution at a con ⁇ centration from about 10 to about 50%.
  • a solution of the bioadhesive protein can be uni ⁇ formly coated on a surface as a primer. Curing of the primer coating occurs in a normal air environment by cross-linking, which may be indicated by the development of a brown or tan color when used in high concentration.
  • a conventional adhesive such as an epoxy adhesive is then applied over the primer coat and the surfaces to be bonded are brought together.
  • an adhesive composition that contains the hydroxylated bioadhesive protein in solution with another adhesive substance.
  • Typical of the adhesives that may be employed in conjunction with the bioadhesive protein of the invention are the carbohydrate adhesives and the synthetic resin adhesives such as the polyacrylates, polyepoxides, resols, etc.
  • the known carbohydrate adhesives that can be employed include chitosan, starch, pectin, glucan, dextran, etc.
  • a preferred carbohydrate adhesive is chitosan purified from crab or shrimp shell chitin by the pro ⁇ cedure of Skujins, J.J. et al.. Arch. Biochem. Biophvs. 111:359 (1965).
  • the free amino groups of chitosan are reactive with the DOPA-derived quinones of oxidized bioadhesive protein, providing covalent cross-links between the two polymers.
  • Chitosan at appropriate concentrations provides bioadhesive protein mixtures with a high viscosity and excellent adhesive strength.
  • the high viscosity is a particularly useful property in underwater applications where diffusion can cause a loss of material before the adhesive has an opportunity to cure.
  • a preferred adhesive mixture comprises from 2% to 30% of the hydroxylated bioadhesive polymer and from 1% to 7% chitosan, the balance being solvent.
  • the pH of the composition is from about 5.5 to 7.0.
  • the composi ⁇ tion can be cured at pH 6.0 by the addition of catechol oxidase or tyrosinase which catalyzes the formation of DOPA-derived quinones and cross-linking.
  • an adhesive composition in which the bioa ⁇ dhesive protein is admixed with other proteins that improve its physical properties such as cohesivity.
  • the mussel adhesive protein is found closely associated with collagen in nature, thus a preferred protein for adhesive composition is collagen.
  • a preferred composi ⁇ tion comprises a solution having 10% to 70% solids, the solids in the solution comprising from 1% to 50% bio ⁇ adhesive protein and from 50% to 99% collagen.
  • the bioadhesive protein of the invention is particularly useful as a biomedical adhesive or sealant, for example, in wound healing. Being a biological material, the bioadhesive protein presents a greatly reduced risk, of toxic degradation products as compared with a chemical synthetic adhesive.
  • the bioadhesive protein can be applied as a biomedical sealant in much the same manner as fibrin (see, e.g., Redl, A., and Schlag, C. , Facial Plasic Surgery 24:315-321 (1985)).
  • the method employed for the synthesis of oligodeoxyribonucleotides is the methyl- phosphite solid-phase method (Matteucci, M.D. and Caruthers, M.H., Tetrahedron Letters. 1:719-722 [1980]) using an automated solid-phase DNA synthesizer manu ⁇ factured by Applied Biosystems, Inc.
  • the starting materials such as the four appropriately protected 5'-dimethoxytrityl-2 *-deoxyribonucleoside-3*-phosphor- amidites as well as the solid support such as silica and controlled pore glass (CPG) (Adams, S.P., Kavka, K.S., Wykes, E.J., Holder, S.B. and Gallappi, G.R., j. Amer. Chem. Soc.. 105:661-663 [1983]) derivatized with appropriately protected 5 ' -dimethoxytrityl- 2'-deoxyribonucleosides, are commercially available.
  • CPG controlled pore glass
  • the DNA synthesis proceeds from the 3'-end to the 5 » -end.
  • the DNA synthesis proceeds from the 3'-end to the 5 » -end.
  • the derivatized solid support containing approximately 1 umol of protected 5*-dimethoxytrityl-2*-deoxyadenosine is loaded in a synthesis column and placed into the automated DNA synthesizer.
  • the coupling cycle consists of detritylation of the solid support with 2% trichloro- acetic acid in dichloromethane; washing with anhydrous acetonitrile; simultaneous addition of an appropriately protected 5 ⁇ -dimethoxytrityl-2 • -deoxyribo- nucleoside-3'-phosphoramidate (10 umol) in acetonitrile and tetrazole (30 umol) in acetonitrile, incubation for one minute, capping of unreacted 5'-hydroxyl groups with acetic anhydride and dimethyl-aminopyridine in tetra ⁇ hydrofuran; oxidation with iodine in a mixture of tetra ⁇ hydrofuran, lutidine and water [2:
  • the coupling cycle is repeated until the desired length of DNA is obtained.
  • the DNA is then partially deprotected by the treatment with thiophenoxide in dioxane/triethylamine and it is released from the solid support by several (2-4) brief treatments (5-10 minutes) with concentrated .ammonium hydroxide. Completely deprotected DNA is obtained by heating the concentrated ammonium hydroxide solution at 60-65 degrees C for 8-14 hours.
  • the DNA is then purified by ion-exchange and linear preparative polyacrylamide gel electrophoresis. The purified DNA is enzymatically phosphorylated at the 5'- end and characterized prior to subsequent ligation.
  • the synthetic dsDNA coding for each of the repeat ⁇ ing decapeptide unit of sequence Ala-Lys-Pro-Ser-Tyr- Pro-Pro-Thr-Tyr-Lys can be selected from sequences in which the coding strand has the formula
  • G, A, T and C represent deoxyribonucleotides containing the bases guanine, adenine, thymine and cytosine, respectively;
  • R represents a deoxyribonucleo ⁇ tide containing guanine or adenine;
  • Y represents a deoxyribonucleotide containing cytosine or thymine; and
  • N represents G, A, T or C.
  • the follow ⁇ ing five double-stranded oligodeoxyribonucleotide sequences are used in preparing the dsDNA insert encod ⁇ ing the bioadhesive precursor protein analog.
  • oligodeoxyribonueleotides were selected with a view toward minimizing repeated DNA sequences which might lead to deletion or recombination by the host.
  • the five oligodeoxyribonueleotides are ligated to each other randomly in order to produce a sequence coding for various repeats of the decapeptide.
  • the automated DNA synthesizer is employed to syn ⁇ thesize the ten single-stranded oligodeoxyribonueleo ⁇ tides having the sequences shown above. There are also synthesized two single-stranded oligodeoxyribonueleo ⁇ tides which can be annealed to form the following blunt-ended linker fragment for the 5* end of the dsDNA insert.
  • the 5' ends of the single-stranded oligodeoxyribonueleotides labeled with asterisks are phosphorylated by treatment with adenosine triphosphate in the presence of polynucleotide kinase.
  • the ten oligodeoxyribonueleotides (1.6 ug each) representing the decapeptide and the four linker oligodeoxyribonueleotides (0.4 ug each) are annealed and ligated in the presence of T4 DNA ligase.
  • the ligation reaction produces a family of blunt-ended dsDNAs of varying length having an average of about 20 of the decapeptide-encoding fragments, ligated in random order, preceded and followed by the 5' and 3' linker fragments, respectively.
  • the synthetic dsDNA thus produced encodes a series of repeated decapeptides directly preceded by the sequence Leu-Glu-Gly-Ser-Met, encoded by the 5' linker and followed by the dipeptide Ala-Lys, encoded by the GCGAAA sequence preceding the stop codon (TGA) in the 3' linker.
  • the synthetic dsDNA from the ligation reaction is run on a 6% polyacrylamide gel according to the procedure of Maniatis, et al. (Biochemistry. .14:3787-3794 [1975]). The band corresponding in size to 20 decapeptide-encoding sequences is cut from the gel and the dsDNA is electroeluted from the gel.
  • the ratio of oligo- deoxyribonucleotide fragments representing the decapept ⁇ ide to linker fragments in the ligation mixture is adjusted proportionately and the dsDNA fragment of desired size is isolated from the gel.
  • this plasmid contains the trpB and trpA regions of the tryptophan synthetase operon under the control of a tac (hybrid trp/lac) promoter.
  • the plasmid has been deposited, in an E_j. coli host (strain GX1668) , at the American Type Culture Collection, Rockville, Maryland, with accession number ATCC 39388.
  • the plasmid (1 ug) is cleaved by treatment with Hoal (1 unit) .
  • Cleavage occurs within the trpB region, leaving 1122 base pairs at the 5' end of the trpB gene linked to the tac promoter.
  • the linearized plasmid has blunt ends.
  • the dsDNA insert containing the decapeptide- encoding sequences (0.2 ug) is blunt-end ligated to the linearized pGX2213 using T4 DNA ligase.
  • the recircularized plasmids are used to transform E. coli strain JM109.
  • This host strain is commercially available, e.g., from P-L Biochemicals, Inc., Bethesda Research Laboratories, Inc. and New England BioLabs, Inc. It contains the lad*? gene which overproduces the lac repressor protein which regulates expression from the tac promoter. Expression from the tac promoter can b e induced by the additi on o f isopropyl—D-thiogalactoside (IPTG) .
  • IPTG opropyl—D-thiogalactoside
  • the host is also recA-. which reduces the likelihood of recombination of repeated decapeptide-encoding sequences in the dsDNA insert. The transformed £.
  • coli JM109 cells are inoculated onto LB-agar plates containing ampieillin and grown for 24 hours. Plasmid DNA prepared from the resultant colonies is screened- by restriction analysis to isolate clones containing a single dsDNA insert in the proper orientation.
  • the isolated clones contain a fused gene which codes for a protein containing the first 374 amino acids of the trpB gene product fused to the five amino acids coded for by the linker at the 5' end of the synthetic dsDNA insert (Leu-Glu-Gly-Ser-Met) , followed by twenty repeats of the decapeptide sequence of the insert and terminating with Ala-Lys, which is coded for by the portion of the linker at the 3' end of the synthetic insert preceding the stop codon.
  • Transformants containing the single dsDNA insert in the proper orientation are inoculated into 2-liter culture flasks containing Luria broth and ampicillin and are grown to mid-log phase (OD600 0.5). IPTG (0.25 mM, final concentration) is added to induce expression. After 8-16 hours, the fusion protein is expressed at high levels in the host cells. The cells are harvested by centrifugation, lysed by sonication, and the fusion protein is recovered by conventional protein recovery techniques.
  • the fusion protein is treated with cyanogen bromide, which cleaves the protein on the carboxyl side of the methionine residue immediately preceding the first decapeptide sequence, thereby separating the bioadhesive precursor protein from the N-terminal frag ⁇ ment of the trpB gene product and the linker-derived peptide fragment.
  • the bioadhesive precursor protein is then isolated by conventional procedures.
  • the following double-stranded oligodeoxyribonucleotide sequence is used in preparing the dsDNA insert encoding the bio ⁇ adhesive precursor protein analog.
  • the automated DNA synthesizer is used to synthesize the following oligodeoxyribonueleotides: A 5' GCT AAG CCA TCT TAC CCA CCA ACC TAC AAG B 5' CTT AGC CTT GTA GGT TGG TGG GTA AGA TGG C 5 » GAA TTC GTC GAC ATG D 5* CTT AGC CAT GTC GAC GAA TTC E 5 « GCT AAG TAA GCT TGG ATC C F 5* GGA TCC AAG CTT A
  • Oligodeoxyribonueleotides A, B, D and E are phosphorylated at the 5* ends by treatment with adenosine triphosphate in the presence of polynucleotide kinase.
  • the oligodeoxyribonueleotides are annealed and ligated in the presence of T4 DNA ligase at a ratio of 4A:4B:1C:1D:1E:1F to produce a dsDNA insert comprising a 5• linker (C and D) , a 3 ⁇ linker (E and F) and a repeated decapeptide coding sequence (A and B) which contains the preferred codons for expression in S.
  • the 5 « linker contains EcoRI and Sail cleavage sites.
  • the 3 f linker contains Hindlll and BamHI cleavage sites.
  • the ligation product is run on a 6% polyacrylamide gel according to the procedure of Maniatis et al., supra.
  • the band corresponding to a dsDNA having approximately 20 repeats of the decapeptide coding sequence is cut from the gel and dsDNA is electroeluted from the gel.
  • the isolated dsDNA fragment is digested with Sail and Hindlll to generate staggered ends.
  • the dsDNA fragment thus produced is inserted into YpGXl, which is represented in Figure 5.
  • YpGXl has unique Sail and Hindlll restriction sites within the tetracycline resistance gene.
  • the plasmid (2 ug) is digested with Sail and Hindlll and the resulting linearized plasmid is ligated with the dsDNA insert in the presence of T4 DNA ligase.
  • the recircularized plasmid is used to transform, £.
  • Plasmids from the Ap ⁇ Tc 8 colonies are analyzed by restriction endonuclease digestion to identify those plasmids containing the dsDNA insert in the proper orientation.
  • PGK phosphoglycerate kinase
  • YpGX60 is digested with Sail and the 2000-base pair fragment corresponding to the PGK pro ⁇ moter and the- first 229 codons of the structural gene is isolated by gel electrophoresis.
  • the plasmid YpGXl into which the synthetic dsDNA fragment encoding 20 repeats of the decapeptide has been inserted is digested with Sail.
  • the PGK fragment isolated from plasmid YpGX ⁇ O is ligated to the linearized YpGXl containing the 20 deca- peptide coding insert in the presence of T4 DNA ligase.
  • the resulting plasmid is used to transform an S_s_ cerevisiae strain such as D8 or AH22 (ATCC #38626) .
  • the transformants are grown on YNBD solid medium supple ⁇ mented with appropriate nutrients and screened immuno- logically to identify colonies that produce the PGK-ad- hesive fusion protein.
  • the isolated transformants are inoculated into 2-liter flasks containing YNBD + tryptophan and grown overnight at 30°C.
  • the cells are harvested by centrifugation and lysed in a French press.
  • the fusion protein is recovered by conventional protein recovery techniques and treated with cyanogen bromide, which cleaves the protein on the earboxyl side of the methionine residue immediately preceding the first decapeptide sequence, to separate the bioadhesive precursor protein from the N-terminal fragment of PGK and the linker-derived peptide.
  • the bioadhesive pre ⁇ cursor protein is then isolated by conventional proce ⁇ dures.
  • Synthetic decapeptide (1.5 mg) of sequence Ala-Lys- Pro-Ser-Tyr-Pro-Pro-Thr-Tyr-Lys, prepared by the Merrifield solid-state method, was combined with 2.0 mg of bovine serum albumin (BSA) in 1.8 ml phosphate- buffered saline. One percent glutaraldehyde (0.2 ml) was added and the solution was incubated 30 minutes at 22°C. Sodium borohydride was added to a final con ⁇ centration of 0.5 mg/ml and incubation was continued at 22°C for one hour. The solution was then dialyzed against phosphate-buffered saline. Amino acid analysis of the resulting protein indicated 35 moles of peptide were coupled per mole of BSA.
  • BSA bovine serum albumin
  • Rabbits were given intramuscular injections with 100 ug of peptide (BSA coupled) in incomplete Freund's adjuvant. Booster subcutaneous injections using incom ⁇ plete Freund's adjuvant were given subsequently in two- week intervals. Antiserum with high-titer antibody reactive toward the decapeptide as well as M.. edulis bioadhesive precursor protein isolated from mussels or analog proteins produced in microorganisms was obtained by this method.
  • Plasmid pGX2287 (NRRL- B15788) , part of a vector/host system for expression of bovine chymosin, was used as the E. coli cloning and expression vector for bioadhesive precursor protein analog coding sequences.
  • Figure 7 outlines the DNA pieces that were assembled during initial cloning experiments.
  • Synthetic DNA coding for one decapeptide flanked by 5' and 3' linkers was cloned between unique Clal and Sphl endonuclease sites of pGX2287 such that a synthetic tribrid gene was formed, containing a 5* segment of the efficiently expressed trpB gene, the bioadhesive precursor protein analog coding sequence and a 3' region encoding 159 carboxy terminal amino acids of bovine chymosin.
  • This tribrid gene was utilized because the 5' sequence and trpB portion provides efficient transcription and translation initiation, and the chymosin sequence causes the formation of insoluble inclusion bodies in i . coli. Inclusion body formation can lend stability to a foreign protein and provide a convenient method of initial purification.
  • Methionine codons were situated on either side of the bioadhesive precursor protein analog coding sequence, so that the bioadhesive precursor protein can be easily excised from the resulting fusion protein by treatment with cyanogen bromide
  • the synthetic DNA shown in Figure 7 was synthesized as seven oligonucleotides using an Applied Biosystems DNA synthesizer (phosphoramidite chemistry) . These oligonucleotides were designated:
  • oligonucleotides were dissolved at a concentration of 1 delta 280 unit/ml.
  • Oligonucleotides #1876, #1877, and #1892 were phosphorylated individually in reactions with T 4 poly- nucleotide kinase and 1 mM ATP with 20 ul of oligo ⁇ nucleotide solution added in a 50 ul kinase reaction.
  • Oligonucleotides #1545 and #1546 were similarly treated, except they were pooled first at a 1:1 ratio. After the enzyme reaction, the solutions were boiled for two
  • Plasmid pGX2287 DNA (5 ug) was digested with 18 units of Clal endonuclease then extracted with phenol- chloroform, ethanol precipitated and dissolved in 0.01 M Tris-HCl, 0.001 M EDTA (pH 8.0) at 0.25 ug DNA/ul.
  • Ten microliters of the Clal-cut pGX2287 DNA was ligated with 25 ul of the 5' linker in a total volume of 40 ul at 16°C for 11 hours. After ligation, the DNA was phenol- chloroform extracted, ethanol precipitated, then dis ⁇ solved in 1 ml water.
  • the DNA solution was concentrated using a Centricon 30 (Amicon) ultrafiltration unit, then washed two times with 2 ml water and centrifuged at 5,000 RPM for ten minutes. The washed and concentrated DNA, largely free of non-ligated linkers, was ethanol precipitated and dissolved in 10 microliters of water.
  • the ligation mixture was diluted to 150 ul in Sphl endonuclease buffer and digested with Sphl. Ten micro- grams of tRNA was added, the solution was phenol-chloro- form extracted, then ethanol precipitated. The DNA was finally dissolved and diluted to 200 ul in T4 ligase buffer and ligated at 15°C overnight.
  • the ligation was used to transform Ej. coli GX3015 (F ⁇ £EEED102 tna2 recA nadA fchlD-pgll [lambda cI857 BamHI]) using standard procedures. Any other JIL. coli host is suitable that has recA.
  • trpED mutations and has a defective lambda lysogen with the lambda cI857 represser.
  • Cells were grown at 30°C on LB + 100 ug/ml ampicillin or minimal medium containing 0.4% glucose, 0.4% acid hydrolyzed casein (casamino acids, Difco) , and 100 ug/ml ampicillin.
  • One characterized transformant upon heat ⁇ ing to 37°C, produced a protein that reacted with both anti-chymosin antibody and anti-decapeptide antibody (produced in accordance with Example 3) in Western blot experiments (Burnette, W.N., 1981, Anal. Biochem.. 112:195-203) .
  • the plasmid in this transformant was named pGX2346 and DNA sequence analysis demonstrated that the synthetic gene contained a 5' and 3' linker with two internal decapeptide coding segments for a total of three decapeptide coding segments (one of these decapeptides is encoded by the 3' linker, see Figure 7).
  • the 5'-end of the three decapep- tide coding sequence generated by Notl digestion of pGX2346 followed by treatment with DNA polymerase I was ligated to the blunt 3'-end of the three decapeptide coding sequence generated by Nael digestion of second aliquot of pGX2346 (see Figure 2) .
  • the 5', 3' and internal linkers all code for amino acids (ala, thr, pro, ser) that are used in the prototype decapeptide and thus do not disrupt the general characteristics of the translation products.
  • pGX2346 DNA was cut with Notl in a volume of 20 ul.
  • a second 0.5 ug aliquot of pGX2346 DNA was cut with Nael.
  • the DNA solutions were extracted with phenol-chloroform, ethanol precipitated and dissolved in 20 ul of water.
  • the Notl-digested DNA was reacted with T4 DNA polymerase in a 100-ul reaction with 0.25 mM dATP, dGTP, dCTP, dTTP at 37°C for 30 minutes to fill in the Notl-generated single-stranded end.
  • the DNA was extracted, precipitated and dissolved in water.
  • the new plasmid was designated pGX2348. When cells containing pGX2348 are grown at 30°C, then shifted to 37°C, they produce a 27,000 molecular weight protein that reacts with anti-decapeptide antibody, as expected.
  • pGX2354 (10- decapeptide repeats) was used for the construction of pGX2358 (15-decapeptide repeats) and pGX2358 was used for the construction of pGX2365 (20-decapeptide repeats) .
  • pGX2358 and pGX2365 were not determined, they have internal Nael sites and also produce immunoreactive proteins of the expected molecular weights before and after cyanogen bromide cleavage, as shown below. These data are consistent with synthetic genes of the expected structure, i.e., tandem decapeptide coding repeats separated by a tripeptide coding segment.
  • Figure 9 shows the amino acid sequence of the 24,077 molecular weight bioadhesive precursor protein analog produced by cells containing plasmid pGX2365 after cyanogen bromide cleavage.
  • the plasmid series pGX2346 through pGX2365 with three to twenty repeats were constructed to be identical, except for the length of the synthetic bioadhesive precursor protein gene. Therefore, it seems likely that the decreased expression level is associated directly with the increased size of the expressed gene.
  • Plasmid pGX2287 contains the bla gene which encodes beta-lac- tamase, providing ampicillin resistance, as well as the trpED genes that in trytophan-deficient medium comple ⁇ ment the trpED102 deletion in the host GX3015 chromo ⁇ some.
  • Transformed cultures of J . coli GX3015 were grown with 100 ug/ml ampicillin and/or in medium lacking tryptophan.
  • a single colony of GX3015 containing one of the plasmids described in Section C above is picked after growth on minimal salts medium (Miller, J.H., "Experi ⁇ ments in Molecular Genetics,” Cold Spring Harbor Labora ⁇ tory, 1972, p.432) supplemented with 0.4% casamino acids and 0.4% glucose and inoculated into 5 ml of LB medium supplemented with 100 ug/ml ampicillin. After reaching an optical density (A600) of greater than 1.0, 0.4 ml of the culture is inoculated into each of two 250-ml baffled flasks containing 50 ml of LB broth supplemented with 100 ug/ml ampicillin. The two flasks are incubated at 30°C and shaken at 250 RPM for 6.5 to 9 hours.
  • Fermentation is carried out using eight liters of the following initial medium:
  • the following fermentation conditions are main ⁇ tained: pH 7.0 (controlled by 5N NH 4 0H, and IN H 3 P0 4 ) Sparge rate 1 wm Temperature 32°C Agitation rate - 800 r.p.m.
  • the feed solution is prepared as follows:
  • Trace Solution 1 500 ml Trace Solution 2 - 100 ml Trace Solution 3 - 10 ml CaCl 2 .2H 2 0 - 50 ml
  • the feed solution is initially added to the broth in a volume of 180 ml and thereafter as needed to main ⁇ tain the glucose level at 10 g/liter. Feed supplemen ⁇ tation is continued until the A600 reaches 20, at which time the cells are induced to express the tribrid bio ⁇ adhesive precursor protein gene from the hybrid lambda OJ/PR promoter. Induction is effected by raising the temperature to 42°C for one hour to deactivate the temperature-sensitive lambda CI857 repressor protein produced by the defective lambda lysogen in the GX3015 chromosome. The fermentation is continued at 37°C for another 6-8 hours.
  • Plasmid pGX2365 was further manipulated in attempts to increase expression of the 20-repeat protein in E. coli. . in particular to examine the effect of chymosin sequences on expression level and intracellular solubility, two new variants of pGX2365 were prepared as described below.
  • Plasmid pGX2365 has a unique Sphl site at the end of the decapeptide multimer coding sequence, a unique Banll site within the chymosin coding sequence and a unique Bell site at the end of the chymosin coding sequence. Oligonucleotides were synthesized and annealed to yield the linker shown below that could be used for stepwise deletion of chymosin sequences from the gene.
  • the linker was first inserted between the Banll and Bell sites of pGX2365 to create pGX2374.
  • the protein pro ⁇ quiz from pGX2374 has only 61 carboxy-terminal amino acids derived from chymosin plus four linker amino acids.
  • the deletion removed the 98 carboxy-terminal chymosin amino acids, including two of the four cysteines originally present in the chymosin segment.
  • Digestion of pGX2374 with Sphl followed by removal of the small Sphl fragment and recircularization resulted in deletion of the remaining chymosin sequences, leaving only the carboxy-terminal amino acids met-pro-gly-leu encoded by the linker sequence after the decapeptide repeats.
  • This plasmid was designated pGX2375.
  • Bioadhesive precursor protein analog sequences constructed as in Example 4 were incorporated into the yeast expression vector YpGX265GAL4 (ATCC #67233) shown in Figure 3.
  • This veast-E. coli shuttle vector replicates at very high copy number (100-200 copies per cell) in Saccharomyces because it carries the yeast 2 micron replication origin and LEU2-d allele from pJDB207 (Beggs, J.D., Jn Alfred Benzon Symposium 16, Molecular Genetics in Yeast, Von Wettstein, D. et al. eds., Munksgaard, Copenhagen, pp. 383-389 (1981)) .
  • Transcrip ⁇ tion initiation and regulation are determined by a promoter that is a hybrid composed of the MF-alphal transcription initiation site (TIS) coupled with the upstream activation site (UAS) from the GALl-10 regula ⁇ tory region.
  • TIS MF-alphal transcription initiation site
  • UAS upstream activation site
  • the GALl-10 UAS must bind GAX.4 pro ⁇ tein, a positive regulator of yeast galactose genes.
  • the GAL4 gene is also carried on the expression plasmid.
  • plasmid YpGX265GAL4 contains a signal encoding sequence derived from the PH05 gene.
  • a synthetic terminator based on that found in a yeast glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was utilized.
  • Patent Application Serial No. 918,147 was excised with restriction enzymes Hindlll and BamHI and cloned into
  • the bioadhesive precursor protein analog-chymosin hybrid gene with its E. coli regulatory sequences was excised from pGX2365 (see Example 4) using Smal and Bell restriction endonucleases and .was joined at the EcoRV and BamHI sites in the yeast expression module in MGX436 to generate MGX441.
  • the E. coli sequences except for the five trpB codons and the methionine codon directly preceding the analog sequence were deleted by oligo- nucleotide-directed mutagenesis using the following oligonucleotide sequence:
  • This generated vector MGX448 This generated vector MGX448.
  • yeast expression module-bioadhesive precursor protein analog-chymosin sequence was excised from the MGX448 vector using S al and Xhol restriction endo ⁇ nucleases and transferred to the yeast-E. coli shuttle vector to generate YpGX277.
  • the GAL4 gene was then added to YpGX277 as a Hindlll fragment at the unique Hindlll site to generate YpGX277GAL4.
  • the bioadhesive precursor protein analog encoded by this plasmid has the sequence PH05 signal-leu-arg-gln-pro-ser-met-ala- ala [ (ala-lys-pro-ser-tyr-pro-pro-thr-tyr-lys)5thr-pro- ala] 4 -ser-met-chymosin(159 amino acids).
  • pGX2365 was digested with Clal and Bell and the fragment containing the bioadhesive precursor protein analog sequence was gel purified.
  • two oligonucleo-tides of sequence 5'ATCAAT and 5' CGATTTGAT were synthesized and annealed.
  • MGX436 double-stranded DNA was digested with EcoRV and BamHI and the large vector fragment was gel purified.
  • the two purified DNA fragments and the annealed oligonucleotides were ligated and E. coli was trans ⁇ formed using standard protocols. Only one plaque con ⁇ tained DNA which digested with BamHI and EcoRV to release a DNA fragment.
  • the bioadhesive precursor protein encoded by YpGX275GAL4 is PH05 signal- asp-ile-lys-ser-met-ala-ala-[(ala-lys-pro-ser-tyr-pro- pro-thr-tyr-lys) 5 thr-pro-ala] 6 -ser-met-chymosin(159 amino acids) .
  • a fragment containing the yeast promoter, signal sequence and bioadhesive precursor protein analog sequence was excised from YpGX277GAL4 using the Hindlll site at the start of the promoter and the Sphl site separating the bioadhesive precursor analog sequence from the chymosin sequence. This fragment was cloned into M13mpl8 such that the Sphl site is adjacent to a BamHI site.
  • the fragment containing the yeast promoter- signal sequence bioadhesive precursor protein analog sequence was then excised from the double-stranded M13 vector using the Hindlll site at the start of the pro ⁇ moter region and the BamHI site from M13. This fragment was then ligated with the yeast E. coli shuttle vector and the GAL4 gene was added as a Hindlll fragment to generate an expression vector, YpGX279GAL4 which is equivalent to YpGX265GAL4 but with the bioadhesive precursor protein analog sequence positioned between the EcoRV site at the end of the PH05 signal encoding sequence and the BamHI site preceding the GAPDH ter ⁇ minator. In the same manner the chymosin coding region was deleted from YpGX275GAL4 to generate YPGX283GAL4.
  • YpGX279 was digested with enzymes Notl and BamHI and the small fragment encoding 20-decapeptide repeats was gel purified.
  • YpGX275 was digested with Notl and BamHI and the large vector fragment carrying the yeast expression module and replication sequences was gel purified. These two fragment were then ligated and E. coli was transformed the resulting vector YpGX284 was isolated.
  • the GAL4 gene was added to YpGX284 as a Hindlll site.
  • the bioadhesive precursor analog protein encoded by YpGX284GAL4 is
  • yeast strain D8 transr formed with expression vectors YpGX275GAL4, YpGX277GAL4, YpGX279GAL4, YpGX283GAL4, and YpGX284GAL4 were first grown on YNBD solid medium (0.7% yeast nitrogen base, 10% glucose, 2% agar) and were then inoculated into 10 ml YPD (1% yeast extract, 2% peptone, 2% glucose) so that the initial A600 reading was 0.1 and were then grown at 28°C with shaking for 17-24 hours.
  • YNBD solid medium (0.7% yeast nitrogen base, 10% glucose, 2% agar
  • 10 ml YPD 1% yeast extract, 2% peptone, 2% glucose
  • the cells were harvested and washed with 10ml YPGal (1% yeast extract, 2% peptone, 2% galactose) and resuspended in an equal volume of YPGal and induced for 6-28 hours.
  • YPGal 1% yeast extract, 2% peptone, 2% galactose
  • T 25 E 125 PH 8.4 buffer 25mM Tris-HCl, 125 mM EDTA, pH 8.4
  • the cells were then harvested, resuspended in 100 25 E 125 buffer, and broken by vortexing in the presence of glass beads. Following the addition of 200 ul T 5 E ⁇ 2 5, the cell lysate was removed from the glass beads and cell debris was pelleted in a microfuge for five minutes.
  • the cells were propagated in the Production medium for approximately 40-45 hours at which time an optical density of about 50-55 was attained,
  • the fermentation conditions were:
  • the cells were then collected by centrifugation and re ⁇ suspended in lysis buffer.
  • linkers were designed to allow production of in-frame fusions with the trpB gene of pGX2346 or related plasmids.
  • the sequences of the linkers are:
  • the 5' linker contains a Notl recognition site and the 3• linker contains a ASP718 recognition site. There ⁇ fore, if one batch of plasmid is digested with Notl and -54-
  • a second batch is digested with ASP178 followed by DNA polymerase I treatment to fill in the 5' overhangs, the DNAs can be ligated to create a new doubled gene as shown below:
  • the oligonucleotides were synthesized on an Applied Biosystems automated DNA synthesizer using phosphora- idite chemistry. Each oligonucleotide was dissolved in H 0 at a concentration of 1.0 A 260 unit/ml. All the oligonucleotides except 2200 and 2199 were phosphor ⁇ ylated with T4 polynucleotide kinase. Twenty ul of a 1.0 O.D./ l solution ( 0.66 ug) of the oligonucleotides was kinased in a 60 ul volume at 37°C for 1.5 hours.
  • the reactions were boiled for two minutes then mixed with their complement, i.e., 2196 with 2197, 2194 with 2195, 2192 with 2193, 2190 with 2191, 2188 with 2189.
  • the phosphorylated oligonucleotide 2201 was mixed with an equal amount of non-phosphorylated 2200 to make the 5' linker and the phosphorylated 2198 was mixed with an equal amount of non-phosphorylated 2199 to make the 3* linker.
  • the samples were heated to boil ⁇ ing, allowed to cool slowly to 20°C and then placed on ice to allow annealing.
  • pGX2287 DNA Ten micrograms of pGX2287 DNA (NRRL-B15788) was digested in a 100 ul volume with 18 units of Clal (Boehringer-Mannheim) .
  • the 10 ug of Clal cut pGX2287 DNA was ligated in a 40 ul volume reaction with 30 ul of the 5' linker solution for 4.5 hours at 15°C.
  • the ligation solution was diluted to 1 ml then concentrated to 50 ul with a Centricon 30 filter (Amicon Corp.). The dilution and reconcentration was repeated two more times to remove non-ligated linkers.
  • the DNA was phenol- chloroform extracted and ethanol precipitated and dis ⁇ solved in 40 ul H 0.
  • a ligation to insert a bioadhesive precursor protein analog gene into pGX2287 with the 5' linker was per ⁇ formed as follows. Approximately 2.5 ug of the modified pGX2287 DNA was ligated in a 40 ul reaction volume reaction containing 4 ul of each of the decapeptide coding oligonucleotide solutions (2194 and 2195, 2192 and 2193, 2190 and 2191, 2188 and 2189) and 2 ul of the hexapeptide coding oligonucleotide solution (2196 and 2197). Two approaches were taken for the addition of 3* linker oligonucleotide (2198 and ' 2199) .
  • oligonucleotide ligation reac ⁇ tions were diluted to a volume of 100 or 150 ul in Sphl digestion buffer and cut with Sphl. The digests were, extracted with phenol-chloroform then ethanol precipi ⁇ tated including the addition of 10 ug tRNA.
  • pGX2385 and pGX2386 Two plasmids that were characterized, (pGX2385 and pGX2386) contain bioadhesive precursor protein inserts of approximately 280 base pairs and 200 base pairs, respectively.
  • the DNA sequence and protein translation of the inserts in pGX2385 and pGX2386 are shown in Figures 17 and 18 respectively. Examination of the DNA sequence of the synthetic genes in pGX2385 and pGX2386 demonstrated that these genes contain the hexapeptide and three of the four decapeptide coding sequences described above. Only decapeptide 4 (oligo 2189 and 2188) is not represented in these two examples.
  • Transformants containing pGX2385 and pGX2386 produced protein that reacted with both anti-chymosin antibody and the anti-bioadhesive precur ⁇ sor protein analog antibody described in Example 3.
  • the linker was designed such that after ligation to join two synthetic genes, the linker creates the deca ⁇ peptide coding sequence shown below between the last decapeptide coding repeat of one original gene and the first decapeptide coding repeat of the second original gene.
  • ..tyr lys gly thr lys ser tyr pro ala ala tyr lys pro lys pro.. ..TAC AAG G GT AC T AAG TCT TAC CC G GCC GCT TAC AAG CCA AAG CCA.. ..ATG TTC C CA TG A TTC AGA ATG GG C CGG CGA ATG TTC GGT TTC GGT..
  • the bioadhesive precursor protein analog gene in pGX2393 was doubled in size by taking advantage of the unique Asp.718 site 3 « of the gene, the unique Notl site 5' of the gene, and the unique Mlul site located 3013 bases upstream of the Notl site.
  • pGX2393 DNA was restricted with Notl and Mlul. and the larger fragments (5.2 kb) was gel-purified.
  • Plasmid pGX2393 DNA was also cut with As.718 and Mlul and the smaller fragment (3.4 kb) was gel-purified. The two fragments were ligated together with oligomers 2220 and 2221 (nonphos- phorylated) which had previously been annealed.
  • E. coli GX1210 (NRRL B-15800) was transformed with the ligation mix.
  • the resulting plasmid YpGX288 should encode 27 decapeptides and six hexapeptides.
  • M13 vector MGX451 was assembled by inserting the bioadhesive precursor analog coding sequence, yeast promoter and PH05 signal encoding sequence from YpGX277GAL4 (Example 6) as a Hindlll-Sphl restriction fragment into Hindlll and Sphl digested M13mpl8. MGX451 was digested with Notl and Sphl and the large fragment was gel purified.
  • YpGX288 (see Example 7) was digested with Notl and Sphl and the small frag ⁇ ment carrying the bioadhesive precursor protein analog coding sequence was gel purified. The two purified fragments were ligated and E. coli was transformed.
  • the desired transformant, having the heterogenous bioa ⁇ dhesive precursor analog coding sequence inserted in the yeast expression module was called MGX456.
  • MGX456 was digested with Notl and BamHI and the small vector frag ⁇ ment was gel purified.
  • YpGX284 was digested with Notl and BamHI and the large vector fragment was gel purified. These two fragments were then ligated and transformed into E. coli.
  • the resulting expression vector was designated YpGX291.
  • the GAL4 gene was then added to the unique Hindlll site of YpGX291 to generate YpGX291GAL4.
  • bioadhesive precursor protein analog coding sequence from plasmid YpGX290 (Example 7) was transferred to a yeast expression vector to generate YpGX297GAL4.
  • a deletion occurred within the bioadhesive pre ⁇ cursor analog protein coding sequence resulting in a sequence encoding a protein of 100,000 rather than 138,000 daltons.
  • Yeast strain D8 was transformed with YpGX291GAL4 and YpGX297GAL4 and the translation products were analyzed as in Example 6. This analysis showed that strain D8 (YpGX291GAL4) produced a bioadhesive precursor protein analog of about 34,000 daltons as expected and this protein composed about 5% of the total yeast cell protein. The same analysis showed that strain D8(YpGX297GAL4) produced a bioadhesive precursor analog protein of about 100,000 daltons and this protein com ⁇ posed about 1-2% of the total yeast cell protein. The size of the bioadhesive precursor protein produced by strain D8 (YpGX297GAL4) was smaller than predicted by the coding sequence present in E. coli vector YpGX290 (138,000 daltons) reflecting the deletion event that occurred during construction of the yeast expression vector.
  • E. coli GX3015 cells containing one of the plasmids described in Example 4 or 7 (32 g wet weight) are suspended in 20 ml 20 mM Tris-HCl, 2 mM EDTA (pH 7.5), 1 mM phenylmethylsulfonyl fluoride, 25 mM iodoacetic acid, and thoroughly disrupted by passage through a French press followed by sonication.
  • the cell debris and inclusion bodies containing bioadhesive precursor pro- tein are pelleted by centrifugation at 27,500 g for 30 minutes at 4°C.
  • the pellet is extensively washed by suspension in 10 mM Tris-HCl, 1 mM EDTA (pH 7.5) and centrifugation. Washing is continued until the super ⁇ natant is clear. The pellet is then dissolved in 15 ml of 6 M guanidine hydrochloride, 5% beta-mercaptoethanol,
  • the bioadhesive precursor pro ⁇ tein is eluted with either a salt gradient (0 to 0.5 M KC1) or a pH change (pH 8 to pH 10) in the buffer.
  • the fractions are assayed by measurement of absorbance at 280 nm and by SDS polyacrylamide gel electrophoresis using both Coomassie blue protein stain and the Western blot assay with specific antibodies (Example 3) .
  • the fractions containing the bioadhesive precursor protein analog are pooled and dialyzed overnight twice against 2 liters of deionized water. The resultant suspension is lyophilized and 1 mg of purified material is obtained. Material could be further purified, if necessary, using -63-
  • the purified protein is hydrolyzed in 6 M constant boiling HCl with phenol crystals in vacuo at 105°C for 24 hours.
  • the amino acids in the acid hydrolysate are identified as O-phthaldehyde (OPA) derivatives which are separated on C18 reverse-phase HPLC column (Fleury, M.O. and D.V. Ashley, Anal. Biochem.. 133:330-335 (1983)).
  • OPA O-phthaldehyde
  • the amin ⁇ acid composition is used to verify purity since only a subset of amino acids is present in bio ⁇ adhesive precursor protein.
  • E. coli cells or yeast cells from a 180 liter fermentation were centrifuged in a Westphalia centri ⁇ fuge, washed with saline and resuspended as 30% solids in 10 mM EDTA, 1.0 mM phenylmethylsulfonyl fluoride (PMSF) , 10 mM iodoacetic acid (IAA) pH 8.0 before break ⁇ ing by a Manton-Gaulin homogenizer. Bioadhesive pre ⁇ cursor protein analog present in an insoluble fraction was collected with the cell debris by Westphalia centri- fugation.
  • PMSF phenylmethylsulfonyl fluoride
  • IAA iodoacetic acid
  • the centrifugation pellet was resuspended to 20% solids in acetic or formic acid at pH 2.2 to 2.5 and mixed for several hours to solubilize the bioadhesive precursor protein analog.
  • the extract was centrifuged or filtered to remove solids and the clear filtrate solution was then adjusted to pH 4 with 5 N KOH in the presence of 10 mM EDTA, 10 mM IAA and 1.0 mM PMSF.
  • A15.1.WP Impurities were removed as a precipitate by filtration or centrifugation.
  • the bioadhesive precursor protein analog in the clear supernatant was then concentrated by ultrafiltration (10,000 M.W. cutoff membrane) and subse ⁇ quently lyophilized.
  • cyanogen bromide 100 g was added and the solution was stirred for 24 hours at room temperature.
  • the reaction mixture was then dried by rotary evaporation and the residue was dissolved in 50-100 ml 6 M guanidine hydro ⁇ chloride at pH 8.0 and centrifuged at 30,000 g for 20 minutes at 4°C.
  • the supernatant was chromatographed on a Sephacryl S-300 column equilibrated with the same solution.
  • the fractions containing bioadhesive precur ⁇ sor protein were collected, adjusted to pH 4.0 with acetic acid, dialyzed against water and lyophilized to recover as a salt-free powder.
  • the pH of the solution is adjusted to 4 with acetic acid and the solution is dialyzed against 100 volumes of 5% acetic acid.
  • the samples are rotatory-evaporated to reduce the volume.
  • the tyrosinase is removed either by using a LH-Sephadex 60 column, which is eluted with 0.2 M acetic acid, or using a membrane filtration method (Amicon PM30, cut off Of 30,000 M.W.) .
  • Microbially produced and hydroxylated bioadhesive protein analog coated onto a surface can be used as a pretreatment or priming substance for conventional adhesives.
  • An example of the use of a bioadhesive protein analog as a primer treatment for bonding two pieces of aluminum is given below.
  • Hydroxylated bioadhesive protein analog prepared as in Example 11 is dissolved in degassed water (optimally at pH 7.0 to 8.0) at a concentration of 10-400 mg/ml (10-40% w/v). The solution is maintained under nitrogen to prevent premature oxidation of DOPA residues to quinones and curing of the adhesive primer.
  • the bioadhesive protein analog solution is sprayed or painted to uniformly moisten an oil-free aluminum surface in a normal air environment.
  • the surface is then dried in a low-humidity environment.
  • a brown or tan color may develop indicating quinone oxidation and chemical cross-linking.
  • the primed surfaces to be bonded are then joined using standard materials such as epoxy glue.
  • an enzyme such as mushroom tyrosinase (Ito et al.. Biochemistry. 222:407-411 (1984)) or Strep- tomvces tyrosinase (Lerch and Ettlinger, Eur. J. Bio ⁇ chem.. 21:427-437 (1972)) is mixed with the non-hydroxy- lated protein (Example 10) immediately prior to applica ⁇ tion (for example, in the nozzle of a spray applicator) at a concentration of 0.01 to 1.0 mg/ml solution. The enzyme under these conditions effects oxidation all the way from tyrosine to the reactive ⁇ uinone species.
  • Blends of bioadhesive protein analogs with other polymers are also used as primers for other adhesives.
  • the hydroxylated bioadhesive protein prepared as in Example 11 is dissolved at a concentration of 30-700 mg/ml (3-70% solids) in water (or a physiological salt solution for medical applications) adjusted to pH 6.0 with dilute acids.
  • a basic solution (approximately 1/50 volume) is added to increase the pH to 8.0.
  • An enzyme such as mussel catechol oxidase (Waite, J.H., J. Mar. Biol. Assoc.. .65:359-371 (1985)) can also be added (final concentra ⁇ tion 0.01-1 mg/ml) in place of or in addition to base solution immediately prior to application to accelerate the oxidation of DOPA residues to quinones to yield more rapid curing.
  • Mixing of components immediately prior to application can occur, for example, in a spray head of a Duploje ⁇ t R syringe as has been described for fibrin sealant (Redl, H. and G.
  • bioadhesive protein is naturally associated with collagen in the byssal threads of the mussels.
  • Collagen is one natural polymer that can be used to increase the cohesive strength of phenolic protein composites.
  • Acid-soluble collagen (Gallop, P.M. and S. Seifter, Methods Enzv ol.. VI:635- 641 (1963)) is dissolved in dilute acid solution at 10- 70% (w/v) .
  • the collagen is mixed with the bioadhesive protein mixtures as described in Example 13 in ratios having from 1% to 50% of the solids comprising bio ⁇ adhesive protein with total solids ranging from 10 to 70%.
  • bioadhesive protein yield more highly cross-linked rigid composites than those with lower percentages of bioadhesive protein.
  • Alkaline solution may be used to neutralize the mixture immediately prior to application. This allows more rapid oxidation and cross-linking (curing) of the mixture. Also, at neutral pH, the collagen will crystallize, providing added cohesive strength.
  • the bioadhesive protein is used in combination with preformed sheets of collagen. This method is analogous to the use of reinforcing steel in cement or graphite fiber in epoxy composites.
  • Collagen sheets such as the commercially available collastat (a Helitrex product distributed by American Home Products Corporation) or other similar products are sprayed or soaked in bioadhesive protein activated as described above in Example 13, then applied to the surface to be bonded.
  • insoluble or crystalline protein sheets can be used as reinforcement for adhesive protein.
  • silk cloth, or sheets formed from solubilized and reprecipitated alpha- keratose from wool keratin fibers J. De Bersagbes, Curr. Probl. Dermatol.. (5:34-86 (1976)
  • polymerized fibrin clot formed from purified fibrinogen, thrombin and Factor VIII Redl, H. and G. Schlag, Facial Plastic Surgery. 2:315-321 (1985)
  • the use of sheets of fibrin may have the additional benefit of helping to promote wound healing (Redl and Schlag, supra) .
  • Chitosan is dissolved in 1% acetic acid to a con ⁇ centration of 30-150 mg/ml (3-15% w/v) .
  • the chitosan solution with a final pH of approximately 6.0 is blended with an hydroxylated bioadhesive protein analog prepared as described in Example 11. Blends typically have bioadhesive protein concentrations between 2 and 30% and chitosan concentrations between 1 and 7%.
  • tyrosinase addition catalyzes the formation of reactive DOPA-derived quinones and cross-linking.
  • Increasing the pH to 8.0 prior to application results in the immediate precipita ⁇ tion of chitosan out of solution which in some cases may not be desirable.

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Abstract

La production recombinante d'analogues de protéines précurseurs bioadhesifs est divulguée. Les analogues de protéines précurseurs bioadhésifs peuvent être hydroxylés et utilisés comme un adhésif dans des environnements humides.
PCT/US1988/000876 1987-03-12 1988-03-11 Production d'analogues de proteines precurseur bioadhesifs par des organismes mis au point genetiquement WO1988007076A1 (fr)

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992010567A1 (fr) * 1990-12-14 1992-06-25 Creative Biomolecules, Inc. Polypeptide bioadhesif synthetique
EP0655497A2 (fr) * 1993-11-03 1995-05-31 Ciba-Geigy Ag Protéase fongique
US5605938A (en) * 1991-05-31 1997-02-25 Gliatech, Inc. Methods and compositions for inhibition of cell invasion and fibrosis using dextran sulfate
US5849537A (en) * 1989-09-19 1998-12-15 Miller Brewing Company Method of expressing antifreeze proteins in yeast
WO2000015789A1 (fr) * 1998-09-09 2000-03-23 United States Surgical Corporation Analogues recombinants de proteines bioadhesives contenant de l'hydroxyproline
WO2006000209A1 (fr) * 2004-06-29 2006-01-05 Jennissen Herbert P Polypeptide lie a un groupe organique
EP1797117A2 (fr) * 2004-08-09 2007-06-20 Battelle Energy Alliance, LLC Clonage et expression de la proteine adhesive recombinante mefp-1 de la moule bleue mytilus edulis
WO2021047648A1 (fr) * 2019-09-14 2021-03-18 Jiangyin Usun Pharmaceutical Co., Ltd. Nouveaux peptides
US11008373B2 (en) 2010-08-20 2021-05-18 Wyeth, Llc. Designer osteogenic proteins
WO2022112177A1 (fr) * 2020-11-26 2022-06-02 Zentraxa Limited Peptides adhésifs

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* Cited by examiner, † Cited by third party
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MX2020011568A (es) * 2018-04-30 2020-11-24 Perfect Day Inc Polimeros de proteina de leche recombinante.

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US4585585A (en) * 1984-03-07 1986-04-29 University Of Connecticut Research & Development Corporation Decapeptides produced from bioadhesive polyphenolic proteins
US4687740A (en) * 1984-03-07 1987-08-18 University Of Connecticut Research & Development Corp. Decapeptides produced from bioadhesive polyphenolic proteins
JPS6485400A (en) * 1987-07-16 1989-03-30 Montefibre Spa Felt and nonwoven fabric based on polyester fiber and glass fiber and its production

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DK163987A (da) * 1986-04-25 1987-10-26 Bio Polymers Inc Fremgangsmaade til fremstilling af dopa-holdige bioklaebende proteiner ud fra tyrosin-holdige proteiner
WO1988003953A1 (fr) * 1986-11-24 1988-06-02 Genex Corporation Bioadhesifs

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US4585585A (en) * 1984-03-07 1986-04-29 University Of Connecticut Research & Development Corporation Decapeptides produced from bioadhesive polyphenolic proteins
US4687740A (en) * 1984-03-07 1987-08-18 University Of Connecticut Research & Development Corp. Decapeptides produced from bioadhesive polyphenolic proteins
JPS6485400A (en) * 1987-07-16 1989-03-30 Montefibre Spa Felt and nonwoven fabric based on polyester fiber and glass fiber and its production

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Biochemistry, issued 10 Sep. 1985, J.H. WAITE et al, "Peptide Repeats in a Mussel Glue Protein: Theme and Variations", see pages 5010-5014. *
Genetic Engineering News, issued April 1985, R. JOHNSON, "Genex Seeks to Clone Mussel Glue Protein", see pages 14 and 18. *
Journal of Comparative Physiology B issued 1986, J.H. WAITE, "Mussel Glue from Mytilus Californianus Conrad: a Comparative Study, see pages 491-496. *
See also references of EP0304486A4 *

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5849537A (en) * 1989-09-19 1998-12-15 Miller Brewing Company Method of expressing antifreeze proteins in yeast
US5928877A (en) * 1989-09-19 1999-07-27 Miller Brewing Company Assay for an antifreeze protein
WO1992010567A1 (fr) * 1990-12-14 1992-06-25 Creative Biomolecules, Inc. Polypeptide bioadhesif synthetique
US5374431A (en) * 1990-12-14 1994-12-20 Creative Biomolecules, Inc. Synthetic bioadhesive
US5605938A (en) * 1991-05-31 1997-02-25 Gliatech, Inc. Methods and compositions for inhibition of cell invasion and fibrosis using dextran sulfate
EP0655497A2 (fr) * 1993-11-03 1995-05-31 Ciba-Geigy Ag Protéase fongique
EP0655497A3 (fr) * 1993-11-03 1997-05-07 Ciba Geigy Ag Protéase fongique.
US5674728A (en) * 1993-11-03 1997-10-07 Novartis Corporation Aspergillus niger vacuolar aspartyl protease
WO2000015789A1 (fr) * 1998-09-09 2000-03-23 United States Surgical Corporation Analogues recombinants de proteines bioadhesives contenant de l'hydroxyproline
WO2006000209A1 (fr) * 2004-06-29 2006-01-05 Jennissen Herbert P Polypeptide lie a un groupe organique
EP1939211A1 (fr) * 2004-06-29 2008-07-02 Morphoplant GmbH Polypeptide lié à un groupe organique
EP1797117A2 (fr) * 2004-08-09 2007-06-20 Battelle Energy Alliance, LLC Clonage et expression de la proteine adhesive recombinante mefp-1 de la moule bleue mytilus edulis
EP1797117A4 (fr) * 2004-08-09 2008-12-31 Battelle Energy Alliance Llc Clonage et expression de la proteine adhesive recombinante mefp-1 de la moule bleue mytilus edulis
US11008373B2 (en) 2010-08-20 2021-05-18 Wyeth, Llc. Designer osteogenic proteins
WO2021047648A1 (fr) * 2019-09-14 2021-03-18 Jiangyin Usun Pharmaceutical Co., Ltd. Nouveaux peptides
CN114375298A (zh) * 2019-09-14 2022-04-19 江阴贝瑞森制药有限公司 新肽
WO2022112177A1 (fr) * 2020-11-26 2022-06-02 Zentraxa Limited Peptides adhésifs

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