Method of producing bioadhesive protein

Vectors are provided that are capable of expressing, in microbial transformants, a protein having the native amino acid sequence of a bioadhesive precursor protein of a marine animal selected from the group consisting of mussels, barnacles, and oysters. The bioadhesive precursor protein can be expressed in transformants, recovered and converted to a bioadhesive protein by hydroxylation.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to the production of bioadhesives that can be 
employed to bond substances for use in wet environments. Typically, the 
bioadhesives of the invention 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. 
2. Brief Description of Background Art 
The properties of adhesives generally must be tailored to meet the 
requirements of the particular environments in which they are to be used. 
Ideally, an 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. 
Many adhesives that exhibit excellent adhesive properties under dry 
conditions suffer a substantial or total loss of those properties in wet 
environments. Furthermore, adhesives of the prior art cannot be cured in 
wet environments. Consequently, it has been particularly difficult to 
develop adhesives for use in wet environments, such as marine adhesives or 
adhesives for use in medical and dental applications. 
Marine mussels and other sessile invertebrates have the ability to secrete 
adhesive substances by which they affix themselves to underwater objects. 
For example, mussels of the genus Mytilus, e.g., the species Mytilus 
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 plaque 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)). While this substance 
might make an excellent adhesive for use in wet environments, isolation of 
the uncured adhesive from mussels for commercial use is not practical, 
since the extraction of 1 kg of the adhesive substance would require the 
use of about 5 to 10 million mussels. Besides involving a very laborious 
process, great care would have to be taken, in case the adhesive were to 
be employed in medical applications, to insure that other mussel proteins 
and contaminants were removed from the bioadhesive prior to use in order 
to prevent antigenic or anaphylactic reactions. 
U.S. Pat. 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. In accordance with the 
disclosure of that patent, 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 subjected to gel 
filtration dialysis to isolate decapeptides of the general formula 
EQU NH.sub.2 
-Ala-Lys-Pro/Hyp-Ser/Thr-Tyr/Dopa-Pro/Hyp-Pro/Hyp-Ser/Thr-Tyr/Dopa-Lys-COO 
H 
The decapeptides produced in this manner are then polymerized 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. 
The procedure of U.S. Pat. No. 4,585,585 still requires the isolation of 
bioadhesive protein from mussel, which, as previously indicated, is 
impractical on a commercial scale. Moreover, in addition to the laborious 
purification procedure, this process adds the additional steps of 
enzymatic digestion, isolation of the decapeptide fragments and chemical 
reassemblage of the fragments into a bioadhesive polymer. This arduous 
procedure is not well-suited to commercial production. Further, the 
polymers produced by this method are quite different from the mussel 
adhesive since they contain only chemically polymerized decapeptides 
produced by trypsin digestion of the natural molecule. Analysis of the 
natural gene described herein demonstrates that there are other 
significant sequence elements in the mussel adhesive from Mytilus edulis. 
Thus a need has continued to exist for means and methods for the efficient 
production of bioadhesives having the excellent properties associated with 
the mussel foot bioadhesive in wet environments. 
A further need has continued to exist for means and methods for producing 
bioadhesives having the properties of the mussel foot adhesive without the 
necessity of handling and processing large quantities of mussels. 
SUMMARY OF THE INVENTION 
This invention involves application of the techniques of recombinant DNA 
technology to the production of bioadhesives of the type produced by 
marine animals such as mussels, barnacles and oysters. In particular, we 
have cloned genes for native bioadhesive precursor proteins (also termed 
polyphenolic proteins) of the mussel Mytilus edulis and constructed 
expression vectors capable of expressing these precursor proteins in 
microbial hosts. The bioadhesive precursor proteins are chemically or 
enzymatically hydroxylated, mimicking a process which occurs in vivo in 
the mussel, to produce a bioadhesive protein which cures to produce an 
adhesive having excellent properties in wet environments. 
In the course of our work, we have isolated multiple cDNA clones coding for 
native M. edulis bioadhesive precursor proteins. Two of the encoded 
proteins (from cDNA isolates 14-1 and 52) have a common C-terminal 
sequence of 134 amino acids, including a hexapeptide followed by twelve 
decapeptides, ten of which display non-homology with the decapeptide 
sequence described in U.S. Pat. No. 4,585,585. 
Another encoded protein, from cDNA isolate 55, has homology with 53 and 110 
amino acids in the proteins encoded by cDNAs 14-1 and 52, as well as 
homology in a different region with 34 and 57 amino acids of the proteins 
encoded by cDNA isolates 56 and 52. The coding homologies are conserved at 
the DNA level. One likely explanation for these observations is that the 
M. edulis polyphenolic protein gene consists of several exons that can be 
spliced together in different combinations at the mRNA level. 
Alternatively, there could be several closely related genes. 
This disclosure is not intended to be limiting to any one exon combination 
or bioadhesive precursor protein gene, as a method is described which 
allows the isolation and use of any existing gene or gene combinations. In 
all the unique decapeptide coding segments observed in the 4 cDNA clones 
described herein, 24 of 39 decapeptides display non-homology with the 
decapeptide described in U.S. Pat. No. 4,585,585. 
Yet a further encoded protein, from cDNA isolate N1, a 2.1 kb clone, 
contains 76 continuous tandem repeats of decapeptides and hexapeptides. Of 
the 63 decapeptides in the N1 protein, 32 display non-homology with the 
decapeptide described in U.S. Pat. No. 4,585,585. 
There is provided, in accordance with this invention, a DNA segment 
comprising codons for the native amino acid sequence of a bioadhesive 
precursor protein of a marine animal, e.g., of a mussel of the genus 
Myutilus, e.g. M. edulis. 
In one embodiment, the bioadhesive precursor protein DNA sequence comprises 
the codons: 
##STR1## 
or a fragment thereof. which code for the amino acid sequence: 
##STR2## 
or a fragment thereof. 
In another embodiment of the invention, vectors are provided for expression 
of bioadhesive precursor protein comprising: 
(a) a DNA sequence comprising codons for the native amino acid sequence of 
a bioadhesive precursor protein of a marine animal, e.g., of a mussel, 
e.g., a mussel of the genus Mytilus, or fragment thereof; and 
(b) a promoter and transcription initiation signal, operably linked to said 
DNA segment, which are capable of effecting microbial expression of the 
bioadhesive precursor protein. 
In yet another embodiment of the invention, there are provided transformant 
microorganisms capable of expressing bioadhesive precursor proteins that 
can be chemically or enzymatically converted to a bioadhesive. The 
transformants are produced by transforming a host microorganism such as an 
E. coli or Saccharomyces cerevisiae with the aforementioned expression 
vector. 
In another embodiment of the invention, there is provided a method of 
producing a bioadhesive protein. A transformant microorganism containing 
the expression vector for the bioadhesive precursor protein is cultured 
and subjected to conditions under which the precursor protein is expressed 
and the precursor protein is recovered. The precursor protein, as will be 
described in detail below, contains amino acid sequence subunits which 
possess tyrosine and proline residues. The precursor protein is converted 
into a bioadhesive protein by chemical or enzymatic hydroxylation, which 
converts at least a portion of the tyrosine residues to 
3,4-dihydroxyphenylalanine (DOPA) residues and, optionally, at least a 
portion of the proline residues to hydroxyproline residues. In a preferred 
embodiment of the invention, the bioadhesive precursor protein is 
hydroxylated by enzymatic treatment, for example, with mushroom 
tyrosinase. 
There are also provided by the invention methods of bonding surfaces which 
involve application of the bioadhesive protein, either as an adhesive per 
se or as a primer coating for an adhesive, to the surfaces being bonded. 
There are also provided by the invention methods for bonding or wetting 
surfaces with composite materials including bioadhesive protein and other 
polymers and/or adhesives.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The preparation of an expression vector of the invention generally involved 
the following series of steps: 
(a) Isolating mRNA from the marine animal, e.g., from the phenol gland of 
the foot of M. edulis; 
(b) Synthesizing complementary DNA (cDNA) and inserting the cDNA into 
bacteriophage; 
(c) Transfecting E. coli with the recombinant bacteriophage and culturing 
the transfected E. coli; 
(d) Selecting clones containing bioadhesive precursor protein genes by 
hybridization of transformants with a radiolabeled probe; 
(e) Insertion of a DNA segment coding for a bioadhesive precursor protein 
into an expression vector as the 3' portion of a gene coding for a hybrid 
fusion protein. In some cases, a methionine residue is located on the 
amino terminal side of the amino acid sequence of the bioadhesive 
precursor protein. (By the term "DNA segment" is intended a portion, but 
less than the entirety, of the genome of the marine animal.) 
The mRNA can be isolated from M. edulis by isolating the foot organ 
containing the phenol gland from live mussels; homogenizing the tissue in 
liquid nitrogen; dissolving the frozen tissue in guanidine thiocyanate as 
described by McCandliss, R. et al., Methods Enzymol., 79:51-57 (1981), or 
lysing the frozen tissue in the presence of ribonucleoside-vanadyl 
complexes and extracting the lysed tissue with phenol as described by 
Berger, S. et al., J. Biol. Chem., 255:2955-2961 (1980); and purifying 
poly A-containing RNA by binding on oligo-dT cellulose as described by 
Aviv, H. and Leder, P., Proc. Natl. Acad. Sci. USA, 69:1408-1412 (1972). 
Similar procedures may be used in isolating the mRNA transcript of the 
gene of other marine animals encoding bioadhesive protein. 
The mRNA extracted, i.e., from the phenol glands, is then employed as a 
template to produce cDNA in the presence of reverse transcriptase, dATP, 
dCTP, dGTP and dTTP, according to well-known procedures. Advantageously, a 
radioactive marker such as [alpha-.sup.32 P]dCTP is used to monitor the 
synthesis. The mRNA moiety is then removed using known procedures and a 
second strand of cDNA is synthesized, using the first strand as a 
template, in the presence of E. coli DNA polymerase I (Klenow fragment), 
dATP, dCTP, dGTP and dTTP. As those skilled in the art are aware, the 
double-stranded (ds-cDNA) DNA produced in this manner contains a hairpin 
loop. The single-stranded loop can conveniently be removed by digestion 
with S1 nuclease. 
The ds-cDNA produced in the manner described is then inserted into a 
cloning vector in order to produce a library of clones to screen for the 
presence of the bioadhesive precursor protein gene. We employed the 
bacteriophage lambda gt10 as a cloning vector; however, those skilled in 
the art will be aware that any of numerous other vectors could 
conveniently be employed to the same end. In order to insert the ds-cDNA 
into the EcoRI site of lambda gt10, we first added synthetic 8-base pair 
EcoRI linkers to both ends of the ds-cDNA after blunting the ends of the 
ds-cDNA in the presence of DNA polymerase I (Klenow fragment) and dATP, 
dCTP, dGTP, and dTTP. To prevent cleavage of the ds-cDNA by EcoRI it was 
first methylated with EcoRI methylase. The linkers were phosphorylated in 
polynucleotide kinase buffer and ligated to the ends of the blunted 
ds-cDNA in the presence of T4 ligase. Multiple linkers were then removed 
from the ends by digestion with EcoRI. The ds-cDNA with EcoRI linkers was 
inserted into EcoRI-cut lambda gt10. 
The cloning vector containing the cDNA is propagated in a suitable host. In 
the case of the recombinant lambda gt10, we packaged the recombinant phage 
DNA into bacteriophage lambda heads for introduction into E. coli strain 
BNN102 (Huynh, T.V. et al., Constructing and screening cDNA libraries in 
lambda gt10 and lambda gt11, In:D. M. Glover (ed.) DNA cloning, Vol I, IRL 
Press, Oxford (1985), pp. 49-78) by procedures described by Enquist and 
Sternberg, Methods Enzymol , 68:281-298 (1979). On this host strain, only 
lambda gt10 bacteriophage-carrying inserts at the EcoRI site will form 
plaques. The transfected hosts are cultured under appropriate conditions 
and examined for plaques. 
The ds-cDNA library produced in the above manner is screened for the 
presence of the bioadhesive precursor protein gene by hybridization with a 
radio-labeled synthetic oligonucleotide probe. One predominant decapeptide 
in the bioadhesive precursor protein is known to be 
ala-lys-pro-ser-tyr-pro-pro-thr-tyr-lys. To screen the recombinant phage 
library for the precursor protein gene, we constructed and radiolabeled 
two 30-base synthetic oligonucleotide probes coding for the decapeptide, 
these two selected randomly from the 98,304 possible coding combinations. 
The two sequences selected were 
EQU GCG AAA CCA AGT TAC CCA CCG ACC TAC AAA 
EQU and 
EQU GCG AAA CCT TCT TAT CCG CCT ACC TAT AAG. 
Using hybridization washing conditions with decreased stringency that were 
previously established in Southern blot experiments and which are 
described in detail in the examples which follow, the radiolabeled probes 
were used to screen plaques which had been fixed on duplicate 
nitrocellulose filter replicates. To our knowledge, the 30-base probes are 
the shortest synthetic probes that have been used successfully in this 
type of screening. Plaques that gave positive signals by autoradiography 
of duplicate filters were purified, screened again and grown as plate 
lysates for DNA preparation as described in Maniatis, T. et al., 
"Molecular Cloning: A Laboratory Manual," Cold Spring Harbor Laboratory, 
pp. 371-372 (1982). The DNA sequence of the isolated clones can be 
determined by well-known methods. 
Using the procedures described generally above and described in greater 
detail in the examples that follow, we isolated several clones. Five have 
been characterized in detail and are identified herein as clones 14-1, 52, 
55, 56, and N1, which contained DNA sequences coding for bioadhesive 
precursor proteins. FIG. 1 presents the DNA sequence and the translated 
amino acid sequence of the insert in clone 14-1. The DNA segment 
comprising the insert in clone 14-1 contains codons for a 203-amino acid 
sequence. In FIG. 1, the sequence has been divided to show 20 subunits, 
including 19 decapeptides and 1 hexapeptide (subunit no. 8). 
FIG. 2 presents the DNA sequence and the translated amino acid sequence 
from the insert in cDNA clone 52. This DNA segment in the insert in cDNA 
clone 52 contains codons for a 334-amino acid sequence. In FIG. 2, the 
sequence has been divided to show 26 subunits which include 23 
decapeptides and 3 hexapeptides, and an amino-terminal proline-rich 
segment. This cDNA clone and two of the other analyzed clones (55, 56) 
contain some noncoding DNA sequence (not shown) which we believe is 
derived from intron sequences. This suggests that some of the clones were 
derived from incompletely processed nuclear RNA 
The DNA sequences of the cDNA clones 14-1 and 52 display extensive homology 
at their 3'-ends In particular, the last 138 codons of the translated 
regions are identical and include codons for a hexapeptide followed by 12 
decapeptides (beginning at codon 75 of clone 14-1 in FIG. 1 and codon 205 
of clone 52 in FIG. 2). Clone 52 contains a segment at the 5'-end that 
codes for a proline-rich segment of 82 amino acids. There is also a direct 
repeat of DNA sequences in clone 52. Specifically, the DNA for codons 
92-147 is exactly repeated in codons 148-203. 
DNA sequences for cDNA clones 55 and 56 are shown in FIGS. 3 and 4. Codons 
19-129 of cDNA clone 55 match the codons 148-259 of cDNA clone 52 with one 
exception. Codon 65 of clone 55 is an alanine while the clone 52 
corresponding codon 195 is isoleucine. 
There is also homology between the cDNA of clone 55 (FIG. 3) and the cDNA 
of clone 56 (FIG. 4). The codons 1-33 of clone 55 match codons 54-86 of 
clone 56. Some of the sequences are in the area where clone 55 also 
overlaps with clone 52, and in the direct repeating segment of clone 52. 
The DNA sequence for clone N1 is shown in FIG. 9. Clone N1 does not have a 
perfect overlap with any of clones 14-1, 52, 55 or 56. Clone N1 also does 
not have a characteristic 5'-end sequence and therefore does not, in all 
likelihood, include the N-terminus of the natural gene. It does, however, 
code for a polypeptide with a molecular weight of about 80,000, a 
substantial portion of the natural polypeptide (molecule weight of 
110,000-130,000). 
Thus it can be seen that the cDNA clones are related in a complex way. 
Although there are areas in common, there is also generally divergence, 
suggesting either the existence of multiple genes or varied patterns of 
mRNA splicing for one gene or both. 
As previously indicated, this invention provides expression vectors for 
microbial expression of bioadhesive precursor proteins. The expression 
vectors contain a DNA segment coding for a bioadhesive precursor protein 
such as that of a mussel of the genus Mytilus or a fragment thereof, the 
DNA segment being operably linked to a promoter and transcription 
initiation signals that are capable of effecting microbial expression. In 
one embodiment, the DNA segment contains codons for at least the portion 
of the amino acid sequence comprising subunits 8-20 of clone 14-1 and 
subunits 14-21 of clone 52; that is, amino acids 72-197 of clone 14-1 and 
amino acids 201-326 of clone 52. 
FIGS. 6 and 7 outline one procedure that is employed to produce expression 
vectors of the invention. These expression vectors are capable of 
effecting expression of a bioadhesive precursor protein as a hybrid fusion 
protein which, in some cases, contains a methionine residue just upstream 
of the protein of interest. Since the bioadhesive precursor proteins 
encoded by all of the clones described above except clone N1 contain no 
methionine residues, they can be conveniently separated from the fused 
sequences by treatment with cyanogen bromide, which cleaves the fusion 
protein at methionine residues. As regards the protein encoded by the N1 
clone, cyanogen bromide treatment may cleave at the methionine residue 
found in the second decapeptide, still leaving essentially the entire 
polypeptide intact. As the amino-terminal non-bioadhesive precursor 
protein amino acids will comprise at most roughly 15%, and in many cases 
much less, of the total adhesive protein precursor, removal of those amino 
acids by cyanogen bromide cleavage will not be necessary for all 
applications. 
Referring to FIG. 6, the DNA segment containing the coding region for the 
bioadhesive precursor protein of clones 14-1 and 52 was obtained from the 
lambda gt10 cloning vectors by EcoRI digestion and subcloned into the 
EcoRI site of plasmid pGX2627. The resultant plasmids were designated 
pGX2368 and pGX2377. 
Plasmids pGX2368 and pGX2377 were subjected to oligonucleotide-directed 
mutagenesis (Zoller, M. J. et al., Methods Enzymol., Vol 100B, R. Woo, L. 
Grassa, and K. Moldave (eds.), Academic Press, New York, pp. 468-500 
(1983)) to create BclI sites located at the translation terminators of 
each gene (see FIGS. 1 and 2). Since pGX2627 is a derivative of pGX1066 
(ATCC No. 39955) that contains an M13 origin of replication, the pGX2368 
and pGX2377 derivatives of pGX2627 were isolated in a single-stranded form 
for the oligonucleotide-directed mutagenesis experiments by infection of 
cultures with IR1 or M13 bacteriophage. The new plasmids containing BclI 
sites were designated pGX2380 and pGX2381. 
The DNA segments containing the coding regions for the bioadhesive 
precursor proteins were then inserted into E. coli and yeast expression 
vectors in the proper reading frame. In an E. coli example presented 
below, the cDNA from phage 14-1 located in pGX2380 was cloned as a trpB 
gene fusion starting with an expression vector identified as pGX2287 that 
was designed for bovine chymosin production. Construction of this vector 
is described in detail in copending, commonly assigned U.S. Pat. No. 
4,798,791. Plasmid pGX2287 has been deposited with the USDA Northern 
Regional Research Laboratory, Peoria, Illinois, with accession No. 
NRRL-B15788. It contains the trpB bovine chymosin gene under the control 
of the leftward operator and rightward promoter regions of phage lambda 
(see copending, commonly assigned U.S. application Ser. No. 534,982) and 
an appropriately positioned transcription initiation signal. It also 
contains an ampicillin-resistance gene. 
Plasmid pGX2287 was cut with ClaI, which cleaved the plasmid in trpB 
24-base pairs upstream from the 5'-end of the chymosin coding sequence. A 
116-base pair ClaI-SphI synthetic DNA was then ligated with linearized 
plasmid. The sequence of the synthetic DNA fragment which was ligated to 
the linearized plasmid is given in FIG. 5(b). The ligated DNA was then 
digested with SphI and the large fragment was isolated, resulting in the 
removal of a substantial portion of the chymosin coding region. The 
plasmid was then recircularized with T4 ligase. The resultant plasmid, 
identified pGX2346, contained the synthetic DNA fragment in place of the 
central portion of the chymosin gene. 
Other specific vector constructions for the expression of the bioadhesive 
precursor protein 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 coding region 
as an in-frame fusion with another gene that is under the control of an 
efficient promoter. Preferably, the fusion is constructed such that the 
encoded fusion protein contains a methionine residue just upstream, e.g., 
within about 10 residues, from the bioadhesive precursor protein segment. 
In the specific construction described herein, pGX2383, there is a 
methionine residue 7 amino acids upstream from the bioadhesive precursor 
sequence. The recovered bioadhesive precursor protein can be treated with 
cyanogen bromide, using conditions well-known in the art, to remove 
extraneous amino acid sequences. As those skilled in the art are aware, 
cyanogen bromide cleaves proteins at methionine residues. Since there are 
no internal methionine residues within the bioadhesive precursor protein 
itself, the protein remains intact. 
It will be readily apparent that modifications can be made in the vector 
construction such that the expression vector can carry the entire coding 
region for the bioadhesive precursor protein or a coding region for a 
fragment thereof. Preferably, the fragment contains at least about 5-20% 
of the coding sequence of a native bioadhesive precursor protein of a 
marine animal, e.g., of a mussel, e.g., of a mussel of the genus Mytilus. 
Encoded amino acids fused to the N-terminal or C-terminal ends of the 
encoded bioadhesive precursor, which cannot be removed by cyanogen bromide 
cleavage, are not believed to interfere with the functioning of the 
bioadhesive protein. 
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, E. coli or other related 
gram-negative organisms such as Salmonella, Klebsiella, Erwinia, etc. With 
plasmids related to pGX2383 the host preferably contains the lambda cI857 
gene coding for temperature-sensitive lambda repressor. In the case of 
pGX2383, we chose to employ E. coli strain GX3015 as the host. This was 
because pGX2383 contains the trpED portion deleted in the trpED102 
chromosomal deletion in GX3015. The plasmid is therefore stabilized by 
growing the transformant in a tryptophan-deficient medium. Strain GX3015 
was deposited on Sep. 13, 1990 at the American Type Culture Collection, 
Rockville, Md., having accession number 55093. 
In a different embodiment, an expression vector may be used to transform 
yeast, e.g., Saccharomyces cerevisiae. Typical vectors of this type are 
disclosed in U.S. application Ser. No. 918,147, filed on Oct. 14, 1986, 
and having the title "Composite Yeast Vectors," incorporated by reference 
herein. One preferred vector for expression of bioadhesive precursor 
protein in yeast comprises the yeast shuttle vector YpGX265GAL4 (ATCC 
#67233). This vector is characterized by a promoter that is a 
hybrid-derived from the S. cerevisiae GALl and MF-alpha 1(alpha-factor) 
promoters. This promoter system permits galactose-regulated expression. 
The regulatory gene comprises the GAL4 gene which encodes GAL4 protein, a 
positive regulator of the GALl-MF-alpha 1 hybrid promoter derived from S. 
cerevisiae. The terminator in the YpGX265GAL4 vector system is derived 
from synthetic DNA and is based on 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 pAT153, and an E. coli selectable marker 
which is ampicillin resistance, also derived from pAT153. The heterologous 
gene (the gene coding for bioadhesive precursor protein) is inserted 
between the PH05 signal-encoding sequence and the GAPDH terminator. 
A typical shuttle vector preparation is as follows. YpGX265GAL4 is digested 
with restriction endonuclease HindIII and large and small DNA fragments 
are gel-purified. The larger fragment is digested with restriction 
endonuclease SmaI, generating two DNA fragments. M13mp9 (commercially 
available) is digested with SmaI and HindIII and treated with calf 
alkaline phosphatase. The DNA fragments created by the SmaI digestion of 
the larger HindIII fragment of YpGX265GAL4 and from digestion of the 
M13mp9 are ligated and used for transformation of E. coli. The 
transformants containing phage double-stranded DNA with the yeast 
promoter-terminator cassette are identified, digested with restriction 
endonucleases (EcoRV and BamHI) and the large fragment (approximately 8 
kilobase) is gel-purified. A plasmid containing bioadhesive precursor 
protein cDNA (i.e., pGX2380) is digested with restriction endonuclease 
XbaI, and the single-stranded overhang is filled with the Klenow fragment 
of DNA polymerase. The DNA molecule resulting from the XbaI and DNA 
polymerase treatment of pGX2380 is digested with BclI. The resulting 
approximately 625-bp DNA fragment is gel-purified. Oligonucleotide linker 
sequences are annealed and used in a three-way ligation with the DNA 
fragments created by restriction of the phage double-stranded DNA and the 
cDNA-derived DNA molecule with the modified XbaI and BclI end. 
E. coli transformants containing the desired phage DNA molecules are 
identified by restriction endonuclease digestion. This phage DNA may be 
digested with restriction endonucleases (SmaI and HindIII), with the small 
(approximately 1,300 base pair) fragment gel-purified. YpGX265GAL4 is 
digested with SmaI and HindIII and the large fragment is gel-purified. The 
DNA molecules created by the SmaI-HindIII digestions are ligated and used 
to transform E. coli. The desired plasmid may be linearized with 
restriction endonuclease (HindIII) and treated with calf alkaline 
phosphatase. The 3.65-kb GAL4-containing fragment obtained from the 
original restriction digestion may by ligated with the linearized DNA and 
used to transform E. coli. 
Saccharomyces strains carrying mutations in the LEU2 structural gene (e.g., 
AH22 (ATCC #38626)) may be transformed with this plasmid, utilizing 
standard methods. The yeast strain may be grown in an appropriate medium 
(YNBD, containing 0.7% yeast nitrogen base, 2% glucose, 20 mg/liter 
L-histidine) to maintain the plasmid. 
While the above typifies one construction technique for yeast expression, 
it is readily apparent that one with ordinary skill can impart 
modifications and variations within the general teaching. As with the 
vectors described above, the expression vector may comprise the entire 
coding region for the bioadhesive precursor protein or coding regions for 
fragments thereof. 
For production of the bioadhesive precursor protein, 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 is cultured under conditions suitable for 
growth and expression of the bioadhesive precursor protein gene. After the 
protein has been expressed, it is recovered from the transformant by known 
methods such as by 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 is 
preferably purified to homogeneity or near homogeneity. In the case of a 
fusion protein such as that expressed by pGX2383, the recovered protein 
can be subjected to cyanogen bromide cleavage to remove extraneous peptide 
sequences. 
The recovered bioadhesive precursor protein is converted to a bioadhesive 
by hydroxylation. In particular, it is likely to be necessary to 
hydroxylate at least a portion of the tyrosine or tyrosine and proline 
residues, an event that occurs in vivo in the mussel. Hydroxylation 
converts tyrosine residues to DOPA residues and, optionally, proline 
residues to hydroxy proline residues. The 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 intermolecular cross-linking which cures the 
adhesive and imparts cohesivity. 
Any suitable chemical or enzymatic means for effecting hydroxylation can be 
employed. It is preferred, however, to effect hydroxylation enzymatically 
using an enzyme such as mushroom tyrosinase or Streptomyces antibioticus 
tyrosinase. Enzymatic hydroxylation 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 872:98-103 (1986). 
Preferably, at least about 10% of the tyrosine residues are hydroxylated. 
The mushroom tyrosinase is removed from the protein using known procedures 
such as binding to a LH-Sephadex 60 column followed by elution with 0.2M 
acetic acid or by membrane filtration. The bioadhesive protein can be 
lyophilized for reconstitution as an adhesive formulation at a later date. 
The bioadhesive protein can be employed in the form of a solution in a 
suitable solvent with or without other adhesive substances. 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 concentration of bioadhesive protein in the solution can range from 
very low to very high, depending upon the intended application. 
The bioadhesive protein or a solution or formulation containing the 
bioadhesive protein can be employed as a primer, i.e., a preadhesive film 
or coating to improve the total adhesive bond to a surface. It may also be 
employed as a component in an adhesive system to impart or improve 
water-resistant adhesivity. Another use within the scope of the invention 
includes use as a thin film membrane, or as a component in a thin film or 
membrane, particularly to impart desirable permeability characteristics 
and/or moisture resistance to such film or membrane. Yet another use is as 
a sealant or component of a sealant to prevent moisture penetration. 
A solution of the bioadhesive protein can be uniformly 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. 
In another embodiment of the invention, an adhesive composition is provided 
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 procedure of Skujins, J. J. et al., Arch. Biochem. 
Biophys., 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 0.1% to 30% of the hyroxylated 
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 composition 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. 
In another embodiment of the invention, there is provided an adhesive 
composition in which the bioadhesive protein is admixed with other 
proteins that improve its physical properties such as cohesivity. A 
preferred protein for this protein is collagen. A preferred composition 
comprises a solution having 0.1% to 70% solids, the solids in the solution 
comprising from 1% to 50% bioadhesive protein and from 50% to 99% 
collagen. 
The bioadhesive protein of the invention, with or without the addition of 
other proteins, is particularly useful as a biomedical adhesive or sealant 
in a variety of medical applications, 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 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 Plastic 
Surgery, 24:315-321 (1985)). 
The following examples are intended to illustrate further the practice of 
the invention and are not intented to limit its scope in any way. 
EXAMPLE 1 
Production of Antibody to Bioadhesive Precursor Protein 
Synthetic decapeptide (1.5 mg) with the predominant sequence of M. edulis 
bioadhesive precursor protein (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.degree. C. Sodium borohydride was added to a final 
concentration of 0.5 mg/ml and incubation was continued at 22.degree. 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. 
Rabbits were given intramuscular injections with 100 ug of peptide (BSA 
coupled) incomplete Freund's adjuvant. Booster subcutaneous injections 
using incomplete 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 produced in microorganisms was obtained by this method. 
EXAMPLE 2 
Isolation of M. edulis Bioadhesive Precursor Protein mRNA 
The adductor muscles of live blue mussels (Mytilus edulis) were cut and the 
foot organ containing the phenol gland was amputated, quickly cut in small 
pieces and frozen in liquid nitrogen. The frozen tissue was shattered into 
small pieces by mixing in liquid nitrogen at top speed in a commercial 
Waring blender with a metal container. The pulverized tissue was stored at 
-80.degree. C. until use. 
The frozen tissue was dissolved in a 4M guanidine thiocyanate solution as 
the first step in the RNA isolation procedure (McCandliss, R. et al., 
Methods Enzymol, 79:51-59 (1981)). From 22.1 g wet weight of tissue, 28.3 
mg of total RNA was obtained. Poly A-containing RNA was purified by 
selecting the RNA that would bind to oligo-dT cellulose (Aviv, H. and P. 
Leder, Proc. Natl. Acad. Sci. USA 69:1408-1412 (1972)). Two oligo-dT 
cellulose selections yielded 0.6% of the total RNA (approximately 170 ug) 
that included polyadenylated mRNA. 
As an alternative to the guanidine isolation procedure, a modification of 
the procedure of Berger, S. et al., J. Biol. Chem., 255:2955-2961 (1980) 
was used. Five grams of the frozen mussel tissue above after 
lyophilization were suspended in 100 ml of 10 mM Tris-HCl, pH7.5, 10 mM 
NaCl, 1.5 mM MgCl.sub.2, 0.2% NP-40, and 10 mM ribonucleoside-vanadyl 
complexes (VRC, Bethesda Research Laboratories), 100 ml of phenol were 
added, and the solution was mixed at top speed in a Virtis homogenizer for 
2 min. The suspension was centrifuged to separate the phases and the 
aqueous phase was removed. The organic phase was extracted with 50 ml of 
0.2M sodium acetate, pH5.5, and the aqueous phase was combined with the 
phase earlier recovered. The aqueous phases were extracted three times 
with phenol containing 0.1% 8-quinolinol. The RNA (with some genomic DNA) 
was precipitated with ethanol. Poly A-containing RNA was purified as 
above. From 5 g of tissue, approximately 150 ug of poly A RNA were 
isolated. 
EXAMPLE 3 
Synthesis and Cloning of i M. edulis cDNA 
Stock Solutions and Materials for First Strand cDNA Synthesis 
0.5M Tris-HCl, pH 8.3 
0.25M MgCl.sub.2 
0.05M dATP, pH 7.0 
0.05M dGTP, pH 7.0 
0.05M dCTP, pH 7.0 
0.05M dTTP, pH 7.0 
[alpha-.sup.32 P]dCTP, 400 Ci/mmol, 1 mCi/ml (Amersham),stabilized aqueous 
solution 
0.1M dithiothreitol (DTT) 
Oligo (dT) .sub.12-18 1,000 ug/ml (Collaborative Research, Waltham, Mass.) 
0.1M sodium pyrophosphate 
0.2M disodium ethylenediaminetetraacetate (EDTA), pH 8.0 
RNasin, ribonuclease inhibitor 30 units/ul, (Promega Biotec) 
Avian myeloblastosis virus (AMV) reverse transcriptase, approximately 
10,000 units/ml (obtained from Life Sciences, Inc., St. Petersburg, Fla.) 
All buffers and salt solutions were autoclaved. The other solutions were 
prepared with sterile glass-distilled water and were stored in sterile 
containers. All stock solutions were stored frozen. All enzymes were 
obtained commercially and used according to the manufacturer's 
specifications unless otherwise noted. 
As a template for cDNA synthesis, mRNA prepared by the guanidine method 
described in Example 2 was employed. In order to follow the synthesis, a 
radioactive marker ([alpha-.sup.32 P]dCTP) was used. This allows 
monitoring of all steps by counting Cerenkov radiation, which does not 
result in any loss of sample. For each ug of mRNA, 2 uCi of [alpha-.sup.32 
P]dCTP at a specific activity of 400 Ci/mmol were used. The radioactive 
material was added to a 2X reaction mixture consisting of 0.1M Tris-HCl, 
pH 8.3, 20 mM MgCl.sub.2, 1 mM dATP, 1 mM dCTP, 1 mM dGTP, 1 mM TTP, 20 mM 
DDT, and 5mM sodium pyrophosphate. This solution was kept on ice. To this 
solution was added mRNA (50 ug/ml, final concentration), oligo 
(dT).sub.12-18 (100 ug/ml), RNasin (600 units/ml), AMV reverse 
transcriptase (800 units/ml), and enough water to dilute the 2X mix to 1X. 
After 5 minutes on ice, the reaction mixture was incubated at 46.degree. 
C. for 10 minutes. Following the incubation, EDTA was added to a final 
concentration of 25 mM. The solution was extracted one time with an equal 
volume of phenol:chloroform (1/1; v/v) and the aqueous phase was 
chromatographed on a column of Sephadex G-100 (0.7.times.20cm) 
equilibrated with 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.1 M NaCl. The 
mRNA:cDNA hybrid in the excluded volume was precipitated by addition of 
0.1 volume of 3M sodium acetate and 2 volumes of 95% ethanol (-20.degree. 
C. to -80.degree. C.). In order to remove the mRNA moiety, the pelleted 
hybrid was dissolved in 300 ul 0.1M NaOH and incubated at 70.degree. C. 
for 20 minutes. The solution was cooled on ice and neutralized with 30 ul 
of 1N HCl. The cDNA was precipitated as described above. 
Stock Solutions and Materials for Second Strand cDNA Synthesis 
0.5M potassium phosphate, pH 7.4 
0.25M MgCl.sub.2 
0.1M Dithiothreitol (DTT) 
0.05M dATP, pH 7.0 
0.05M dGTP, pH 7.0 
0.05M dCTP, pH 7.0 
0.05M dTTP, pH 7.0 
E. coli DNA polymerase I (Klenow fragment), 
approximately 5,000 units/ml (Boehringer-Mannheim) 
10X S1 nuclease buffer: 0.5M sodium acetate, pH 4.5; 10mM ZnSO.sub.4, 2M 
NaCl, 5% glycerol. 
It was not necessary to use a radioactive label in the second strand, since 
the first strand was labeled. A 2X reaction mixture consisting of 0.2 M 
potassium phosphate, pH 7.4, 20 mM MgCl.sub.2, 2 mM DTT, 0.4 mM each of 
dATP, dGTP, dCTP, and dTTP was prepared and kept on ice. To this mixture 
was added an aqueous solution of cDNA containing the Klenow fragment of E. 
coli DNA polymerase I (100 units/ml, final concentration), and water was 
added to dilute the reaction mixture to 1X. The solution was incubated 
overnight at 15.degree. C. After the incubation, EDTA was added to 25 mM, 
the solution was extracted once with an equal volume of phenol: chloroform 
(1/1; v/v), and the aqueous phase was chromatographed on a 0.7.times.20 cm 
column of Sephadex G-100 equilibrated with 10 mM Tris-HCl, pH 8.0, 1 mM 
EDTA, and 0.1M NaCl. The DNA in the excluded fractions was precipitated 
with ethanol as described above. 
At this point, the ds-cDNA was in the form of a hairpin. The 
single-stranded loop was removed by digestion with S1 nuclease. The 
ds-cDNA was dissolved in water and 0.1 volume of 10X S1 buffer was added. 
An appropriate amount of S1 nuclease was added and the solution was 
incubated 20 minutes at 37.degree. C. The amount of enzyme added was 
determined empirically for each enzyme preparation, since the activity 
varied from one preparation to another. This was done by measuring the 
decrease in TCA-precipitable counts from the ds-cDNA. Generally, a 
decrease of 20-40% was observed. The S1-digested cDNA was extracted once 
with phenol: chloroform and the cDNA in the aqueous phase was precipitated 
with ethanol as described above. 
Addition of Linkers to cDNA 
For insertion of the cDNA into the EcoRl site of lambda gt10 vector, EcoRI 
linkers were added to the ends of cDNA molecules. To prevent cleavage of 
internal EcoRI sites in the cDNA molecules with EcoRI, the cDNA was first 
methylated with EcoRI methylase. 
Stock Solutions and Materials 
5.times.EcoRI methylase buffer--0.5M Tris-HCl, pH 8.0, 0.05 M EDTA. 
8 mM S-adenosyl methionine (SAM)--solution in 0.01M H.sub.2 SO.sub.4, pH 2, 
10% ethanol. 
1 mg/ml bovine serum albumin (BSA)--solution in water, sterile filtered. 
8-base pair EcoRI linkers--10 A.sub.260 /ml, obtained from Collaborative 
Research, Massachusetts. 
10 mM ATP, pH 7.0 
10.times.T4 polynucleotide kinase buffer--0.7M Tris-HCl, pH 7.6, 0.1M 
MgCl.sub.2, 50 mM dithiothreitol. 
gamma[.sup.32 P]-ATP--10 mCi/ml, 2,000 Ci/mmol, stabilized aqueous 
solution. 
10.times.T4 ligase buffer--0.5 M Tris-HCl, pH 7.8, 0.1M MgCl.sub.2, 0.2M 
dithiothreitol. 
10.times.DNA polymerase buffer.times.0.5M Tris-HCl, pH 7.2, 0.1M 
MgSO.sub.4. 
2 mM dATP, dCTP, dGTP, dTTP mixture, pH 7.0. 
1 mM dithiothreitol 
10.times.EcoRl buffer.times.1.0M Tris-HCl, pH 7.5, 0.5 M NaCl, 0.05M 
MgCl.sub.2. 
The cDNA fragment produced as described above was dissolved in 20 ul of 
5.times.EcoRI methylase buffer, SAM was added to 80 uM, BSA was added to 
0.4 mg/ml, water was added to bring the volume to 99 ul, and 1 ul of EcoRI 
methylase (20,000 units/ml, New England Biolabs) was added. The reaction 
was incubated at 37.degree. C. for 60 minutes. The reaction was then 
extracted 2 times with phenol and ethanol-precipitated. The methylated 
cDNA was collected by centrifugation and dried. 
Before addition of the linkers, the cDNA was treated with DNA polymerase I 
(Klenow fragment) in the presence of deoxynucleoside triphosphates to make 
the ends of the cDNA blunt. The cDNA was dissolved in 24 ul of 1X DNA 
polymerase buffer containing 80 uM each dATP, dCTP, dGTP and dTTP. Two 
units of DNA polymerase (Klenow fragment) were added and the reaction 
mixture was incubated at 23.degree. C. for 10 minutes. EDTA was added to 
20 mM and the cDNA was extracted two times with phenol, once with 
CHCl.sub.3, and ethanol-precipitated. The cDNA, which was EcoRI-methylated 
and blunt-ended, was collected by centrifugation, washed once with cold 
70% ethanol, and dried. 
To prepare the linkers for addition to the cDNA, the linkers first had to 
be phosphorylated. 400 Picomoles of 8-base pair EcoRI linkers were 
phosphorylated in 1X polynucleotide kinase buffer with 20 uCi of 
[gamma-.sup.32 P]-ATP and 5 units of polynucleotide kinase. The reaction 
was incubated at 37.degree. C. for 15 minutes. Unlabeled ATP was then 
added to 1 mM and the reaction was incubated at 37.degree. C. for 30 
minutes. The enzyme was inactivated by heating the reaction at 65.degree. 
C. for 10 minutes. 160 picomoles of the phosphorylated linkers were then 
ligated to the cDNA. The blunt-ended, methylated cDNA was dissolved in 10 
ul of water, 160 picomoles of linkers were added, 10.times.T4 ligase 
buffer was added to 1 1X, ATP was added to 1 mM, and 2 units of T4-DNA 
ligase (Boehringer-Mannheim) were added. The reaction mixture (a total of 
20 ul) was incubated at 15.degree. C. for 16 hours. The ligase was 
inactivated by heating the reaction of 65.degree. C. for 10 minutes. 
At this point, the cDNA had multiple linkers at the ends. Excess linkers 
were removed by digestion with EcoRl. 
The ligation reaction was diluted with 5 ul of 10.times.EcoRl buffer, 5 ul 
of 1 mg/ml BSA and 19 ul of water. Prior to addition of EcoRl, 1 ul of the 
mix was removed for analysis by gel electrophoresis. Ten units of EcoRl 
(New England Biolabs., 10 units/ul) were added and the reaction was 
incubated at 37.degree. C. for 1 hour. The reaction was extracted 1 time 
with phenol and run over a column of Sephadex G-100 equilibrated with 10 
mM Tris-HCl, pH 8.0, 1 mM EDTA, and 0.1M NaCl. The DNA in the excluded 
fractions was concentrated by use of a Centricon 30 microconcentration 
unit (Amicon) according to manufacturer's instructions. The cDNA was 
loaded on 5-25% sucrose gradient in 50 mM Tris-HCl, pH 8.0, 1 mM EDTA and 
the gradients were centrifuged at 5.degree. C. at 38,000 r.p.m. for 17 
hours in a Beckman SW41 rotor. Fractions were collected and were analyzed 
by counting Cerenkov radiation. Parallel gradients were run with molecular 
weight markers Fractions containing cDNA of approximately 1,000-base pairs 
and longer were pooled and the cDNA was concentrated with a Centricon 30 
microconcentration unit. The cDNA was washed four times with 1 ml of 
H.sub.2 O to remove the sucrose and was dried in vacuo. 
Preparation of Recombinant Bacteriophage DNA 
Bacteriophage lambda gt10 DNA (Huynh, T.V. et al., Constructing and 
screening cDNA libraries in lambda gt10 and lambda gt11, In: D. M. Glover 
(ed.) DNA cloning, Vol. 1, IRL Press Oxford 1985, pp. 49-78) 
prepared by standard methods (Maniatis, et al., Molecular Cloning, Cold 
Spring Harbor, 1982) was linearized by digestion with EcoRI, 
phenol-extracted, and precipitated with ethanol. Fifty ng cDNA were mixed 
with 1 ug EcoRI-cut lambda gt10 (a molar ratio of approximately 2:1) in 50 
mM Tris-HCl, pH 7, 10 mM MgCl.sub.2, 20 mM dithiothreitol, and 1 mM ATP, 
and 1 unit of T4-DNA ligase (Boehringer-Mannheim) was added. The ligation 
mixture was incubated for 16 hours at 15.degree. C. A small portion of the 
reaction mix was analyzed by agarose gel electrophoresis. The desired 
product at this point was high molecular weight, concatameric DNA, which 
was in the form required for efficient packaging into empty bacteriophage 
lambda heads. 
Packaging of the Recombinant DNA into Bacteriophage Lambda Heads and 
Transfection into E. coli 
The recombinant DNA was packaged into bacteriophage lambda heads for 
introduction into E. coli, by procedures described by Enquist and 
Sternberg, Methods Enzymol., 68:281-298 (1979). Packaging extracts are 
available commercially (Promega Biotec, Madison, Wis., and other sources) 
and were used according to the manufacturer's instructions. The ligated 
DNA was mixed with the packaging extracts (50 ul), incubated at 23.degree. 
C. for 2 hours; the phages were diluted with 0.5 ml, 10 mM MgSO.sub.4, 10 
mM Tris-HCl, pH 7.5, 0.01% gelatin, and a few drops of chloroform were 
added to the mixture. The packaged phages were stored at 4.degree. C. 
As a host for titration and propagation of the phage, E. coli strain BNN102 
(Huynh, T.V. et al., supra) was used. On this host strain, only lambda 
gt10 bacteriophages carrying inserts at the EcoRI site will form plaques. 
The host strain was grown overnight at 37.degree. C. in LB-broth 
containing 0.2% maltose, to induce synthesis of the phage receptor in the 
host. The cells were collected by centrifugation and resuspended in 
one-half volume of 10 mM MgSO.sub.4. Cells prepared in this manner were 
used for 1-2 days. 
The phage were diluted serially in 10 mM MgSO.sub.4, 10 mM Tris-HCl, pH 
7.5, and 0.01% gelatin. Fifty ul of diluted phage were added to 0.2 ml of 
E. coli cells, and the mixture was incubated at 37.degree. C. for 15 
minutes to allow absorption of the phage. The infected cells were mixed 
with molten (47.degree. C.) LB broth containing 0.7% agar and poured on 
the LB-agar plate. After the top agar hardened, the plates were incubated 
at 37.degree. C. for 5-6 hours, at which time plaques could clearly be 
seen in the lawn of bacteria. A library of approximately 500,000 
recombinant phages was obtained. 
EXAMPLE 4 
Selection of Bioadhesive Precursor Protein cDNA Clones from the M. edulis 
cDNA Phage Library 
The lambda gt10 phage library was plated on twenty 15-cm petri dishes at a 
density of approximately 25,000 phage/plate. Duplicate nitrocellulose 
filter replicates were prepared for hybridization screening (Benton, W. D. 
and R. W. Davis, Science, 196:180 (1977)). Two 30-base synthetic 
oligonucleotides (GCG AAA CCA AGT TAC CCA CCG ACC TAC AAA and GCG AAA CCT 
TCT TAT CCG CCT ACC TAT AAG) were prepared with an Applied Biosystems DNA 
synthesizer. These sequences are two of the possible 98,304 coding 
sequences for the predominant decapeptide 
(ala-lys-pro-ser-tyr-pro-pro-thr-tyr-lys) of M. edulis bioadhesive 
precursor protein. The oligonucleotides were radioactively labeled to a 
specific activity of approximately 10.sup.8 cpm/ug with gamma-[.sup.32 
P]-ATP (New England Nuclear) and T4 polynucleotide kinase 
(Boehringer-Mannheim), then used as hybridization probes. The radioactive 
oligonucleotides (approximately 3.0 ug) were added to 250 ml hybridization 
solution containing 20% formamide, 6XSSC, 5X Denhardt's solution, 50 mM 
phosphate buffer (pH 6.8), 100 ug/ml sonicated denatured salmon sperm DNA 
and 10% dextran sulfate. The filters were hybridized for 14 hours at 30 
.degree. C., then washed five times briefly with 300 ml 6XSSC at 
22.degree. C., one time with 300 ml 1XSSC at 22.degree. C. and once at 
42.degree. C. for 30 minutes with 500 ml 1XSSC. The filters were air-dried 
and autoradiographed at -80.degree. C. with Kodak XAR X-ray film for 12 
hours. 
These hybridization and washing conditions were previously established in 
hybridization to M. edulis DNA with the same probe on Southern blots 
(Southern, E., J. Mol. Biol., 98:503 (1976)). A 42.degree. C. wash in 
1XSSC was the highest temperature that demonstrated apparently specific 
hybridization to a fragment of BamHI-digested M. edulis DNA. The DNA for 
the Southern blot experiment was isolated from the frozen tissue described 
in Example 2 by the method of Blin and Stafford (Blin, N. and D. W. 
Stafford, Nuc. Acids Res., 3:2303-2308 (1976)). 
Plaques giving signals by autoradiography on the duplicate filters were 
purified by picking, diluting, plating and repeating the hybridization 
screen described above. Isolated individual plaques giving radioactive 
signals were picked and grown as plate lysates for DNA preparation as 
described (Maniatis, T. et al., "Molecular Cloning: A Laboratory Manual," 
Cold Spring Harbor Laboratory, 1982, pp. 371-372). 
EXAMPLE 5 
DNA Sequence Analysis to Verify Clones 
DNA from lambda gt10 clone 14-1 and other clones with putative bioadhesive 
precursor protein cDNA inserts were digested with restriction endonuclease 
EcoRI. The EcoRI cDNA fragment was cloned into M13 mp11 (Norrander, J. et 
al., Gene, 26:101-106 (1983)). The bacteriophage M13 derivatives were 
constructed using methods described by Messing (Messing, J., Methods 
Enzymol., 101:20-78 (1983)). Both orientations of the inserts were 
represented in independent clones. Some of the larger inserts were 
progressively shortened by the procedure of Dale et al. (Plasmid, 13:31-40 
(1985)). The phage clones were sequenced by the dideoxy method (Biggin, M. 
D. et al., Proc. Natl. Acad. Sci. USA, 80:3963-3965 (1983)). The recorded 
DNA sequences were analyzed with VAX 780 computer assistance. The DNA 
sequence and translated amino acid sequence of clone 14-1 are presented in 
FIG. 1. Clone 14-1 encodes a polypeptide with tandem repeats of 19 
decapeptides and 1 hexapeptide. Clone 52, presented in FIG. 2, encodes a 
polypeptide with a mix of 22 decapeptide repeats and 4 hexapeptide repeats 
preceded by an 82-amino acid segment that is proline rich. Proline-rich 
segments such as this had not been previously described in M. edulis 
polyphenolic protein. Clones 14-1 and 52 share considerable homology in 
their 3'-ends. After a short untranslated trailer sequence in both clones, 
there is the polyadenylation signal AATAAA located just prior to a stretch 
of A residues. These features are expected at the authentic 3+-terminus of 
mRNA. 
EXAMPLE 6 
Isolation of Full-Length M. edulis Bioadhesive Precursor Protein cDNA 
A. mRNA isolation 
The partial bioadhesive precursor protein cDNAs shown in FIGS. 1-4 that 
were isolated and characterized as described in Examples 1-5 are used to 
isolate full-length bioadhesive precursor protein cDNA. This is 
accomplished through use of the initial clones as hybridization probes of 
more carefully prepared cDNA clone banks. 
The mRNA used in Example 3 was prepared from mussels obtained in a grocery 
store that were not actively producing byssus threads and adhesive 
plaques. This may have resulted in low-level and poor quality adhesive 
protein mRNA. In order to obtain mRNA of better quality, fresh mussels 
were obtained directly by air freight delivery from the Blue Gold Sea 
Farms in Rhode Island. The mussels had no treatment after harvesting other 
than tumbling to clean their outer shells. The mussels were introduced 
into a 20-gallon aquarium tank placed in a 4.degree. C. room with 10 
gallons of synthetic sea water (Instant Ocean, Aquarium Systems, Mentor, 
Ohio) heated to approximately 12.degree.-15.degree. C. The tank water was 
mixed and the temperature regulated with a Precision H&7B heater. 
Additional rapid agitation was provided by a lighting series 30 mixer 
(Mixing Equipment, Co., Inc., Rochester, N.Y. 14603). When individual 
mussels became active in attachment, they were removed and the distal end 
of the foot was quickly amputated and immediately frozen by dropping into 
liquid nitrogen. The tissue was ground into a powder by mixing in liquid 
nitrogen in a Waring blender, and then lyophilized. The mRNA was isolated 
by the procedure using ribonucleoside-vanadyl complexes as described in 
Example 2. 
B. cDNA Synthesis and cDNA Clone Selection 
The mRNA is used to synthesize cDNA as described in Example 3. However, 
this time when the double-stranded cDNA is fractionated on a 5-25% sucrose 
gradient, a much more stringent size selection is used where only cDNA of 
approximately 3,000 base pairs of larger is pooled for cloning. 
The phage library and screening is carried out as described in Examples 3 
and 4, except that the EcoRI insert fragment of clones 14-1 is isolated 
and made radioactive by nick translation (Rigby, P. W. J. et al., J. 
Molec. Biol. 113:237) with [alpha-.sup.32 P]-dCTP label. Hybridization is 
carried out at 42.degree. C. in 6XSSC, 50% formamide, with 5X Denhardt's 
solution and 100 ug/ml denatured salmon sperm DNA. The filters are washed 
at 50.degree. C. with 0.1.times.SSC with 0.01% SDS. Hybridization-positive 
plaques identified in duplicate are purified and DNA is prepared as 
described in Example 4. Clones with the largest insert size are selected 
for DNA sequence anlysis. Clones found with a characteristic 5'-terminal 
sequence and an open reading frame that codes for a protein of 
approximately 120,000 M.W. are full-length bioadhesive precursor protein 
cDNA clones. 
EXAMPLE 7 
Construction of Expression Vector pGX2383 for Microbial Production of M. 
edulis Bioadhesive Precursor Protein 
The steps outlines below and illustrated schematically in FIG. 6 yielded a 
plasmid that expresses M. edulis bioadhesive precursor protein as a hybrid 
fusion protein. The presence of a methionine residue immediately on the 
amino-terminal side of the adhesive protein allows isolation of basically 
pure bioadhesive precursor protein sequence after cyanogen bromide 
cleavage (Gross, E., Methods Enzymol., 11:238 (1967)). Plasmid pGX2287 
(NRRL-B15788), part of a highly optimized vector/host system for 
expression of bovine chymosin (see copending U.S. Pat. application No. 
644,998 and U.S. Pat. No. 4,798,791) was used as the starting vector. 
The EcoRI fragment from lambda gt10 clone 14-1 was subcloned into the EcoRI 
site of plasmid pGX2627. Plasmid pGX2627 is derived from pGX1066 (on 
deposit at the American Type Culture Collection, Rockville, Md., and 
having accession number ATCC 39955). To derive pGX2627, a 514-base pair 
RsaI fragment from wild-type phage M13 containing the M13 origin of 
replication was inserted into the EcoRV site of pGX1066. The DNA sequence 
of pGX2627 is shown in FIG. 7.) This plasmid contains a multi-restriction 
site sequence as shown in FIG. 5(a). Ten micrograms of plasmid pGX2627 DNA 
and 20 micrograms of lambda clone 4-1 DNA were cut with EcoRI (New England 
Biolabs). This enzyme reaction and all those outlined below were performed 
according to the manufacturer's recommendations. The pGX2627 DNA was then 
treated with alkaline phosphatase. After phenol-chloroform extraction and 
ethanol precipitation using standard procedures in the art, a ligation 
using T4 DNA ligase in 20 ul with 1 ug of the EcoRI-cut, 
phosphatase-treated pGX2627 DNA and 12.5 ug EcoRI-treated lambda 14-1 DNA 
was started at 15.degree. C. After 30 minutes, the reaction mixture was 
diluted to 50 ul and ligation was continued for nine hours. The ligation 
mix was used to transform E. coli DHl (D. Hanahan, J. Mol. Biol. 
166:557-580, 1983) using standard procedures (Mandel, M. and Higa, A., J. 
Mol. Biol., 53:154, 1970). Most of the transformants contained the desired 
insert. One recombinant plasmid was identified as pGX2368 as shown in FIG. 
6. 
Plasmid pGX2346 was constructed from pGX2287 by insertion of a synthetic 
oligonucleotide (FIG. 5b) between the ClaI and SphI sites of pGX2287. 
Plasmid pGX2287 DNA was cut with ClaI and ligated with synthetic DNA at 
high molar concentration. The ligated DNAs were then cut with SphI and 
ligated again at low concentration to form circles. Plasmid pGX2346 was 
identified by its decreased size and verified by DNA sequencing. 
An in-frame gene fusion between the trpB portion of pGX2346, and the 
bioadhesive precursor protein cDNA of pGX2368 is constructed in the 
following manner: A BclI endonuclease recognition site was first placed at 
the translation stop codon of pGX2368 by changing two bases as indicated 
in FIG. 1 using oligonucleotide-directed mutagenesis (Zoller, M. J. and M. 
Smith, Methods Enzymol., 100:457-500, 1983) to create plasmid pGX2380. 
Both plasmids pGX2380 and pGX2346 are grown for DNA preparation in an E. 
coli host that contains the dam mutation (defective in DNA adenine 
methylase) so that they could be digested with BclI. The non-methylated 
pGX2346 DNA is cut with NotI and the pGX2380 DNA is cut with XbaI. Then 
both DNAs are treated with E. coli DNA polymerase (Klenow fragment) to 
fill in the 5' single-stranded DNA overhangs. The DNAs are then ligated at 
high DNA concentration (approximately 2 ug of each DNA in 20 ul) with T4 
ligase. The ligation product is cut with BclI then ligated again at low 
DNA concentration (approximately 1 ug total DNA in 150 ul volume) and used 
to transform E. coli GX3015. A transformant with the desired construction 
(see FIG. 6) is designated pGX2383. GX3015 cells with plasmid pGX2383 
produce a bioadhesive precursor protein of approximately 24,000 M.W. upon 
induction of the hybrid lambda promoter by a shift of growth temperature 
from 32.degree. to 37.degree. C. 
EXAMPLE 8 
Construction of Expression Vector pGX2381, for Microbial Production of M. 
edulis Bioadhesive Precursor Protein 
The EcoRI insert fragment from lambda gt11 clone 52 (see FIG. 2) was cloned 
into the EcoRI site of plasmid pGX2627. Plasmid pGX2627 is derived from 
pGX1066 that has been deposited at the American Type Culture Collection, 
Rockville, Md. (ATCC 39955) on Dec. 20, 1984. To derive plasmid pGX2627, a 
514-base pair RsaI fragment from wild-type phage M13 containing the M13 
origin of replication was inserted into the EcoRV site of pGX1066. The DNA 
sequence of pGX2627 is shown in FIG. 7. 
Ten micrograms ms of plasmid pGX2627 DNA and twenty micrograms of lambda 
clone 52 DNA were cut with EcoRI. The pGX2627 DNA was then treated with 
alkaline phosphatase. After phenol-chloroform extraction and ethanol 
precipitation using standard procedures in the art, one microgram of 
phosphatase-treated pGX2627 DNA and twelve micrograms of the EcoRI-cut 
lambda clone 52 DNA were ligated at 15.degree. C. in a twenty microliter 
volume with T4 polynucleotide ligase. After thirty minutes the ligation 
mixture was diluted to fifty microliters with ligation buffer and the 
reaction was continued for nine hours. The ligation mix was used to 
transform E. coli DH1 (D. Hanahan, J. Mol. Biol. 166:557-580, (1983)) 
using standard procedures (M. Mandel and A. Higa, J. Mol. Biol. 53:154, 
(1970)). Most of the transformants contained the desired insert. One 
recombinant plasmid with the cDNA inserted in the orientation depicted in 
FIG. 6 was identified as pGX2377. 
A BclI endonuclease site was placed at the translation stop codon of the 
cDNA in pGX2377 by changing the two bases as indicated in FIG. 2 using 
oligonucleotide-directed mutagenesis (J. M. Zoller and M. Smith, Methods 
Enzymol. 100:457-500, (1983)) to create pGX2381. The mutagenic 
oligonucleotide had the DNA sequence ACAGATATTGATCACAATATTA. 
EXAMPLE 9 
Yeast Expression of Bioadhesive Precursor Protein (Clone 14-1) 
YpGx265GAL4 in E. coli host was deposited on Oct. 9, 1986 at the American 
Type Culture Collection, Rockville, Md., having accession number ATCC 
67233. 
The YpGX265GAL4 vector contains the following functional units: 
Promoter--Hybrid derived from the S. cerevisiae GALl and MF-alpha 1 
(alpha-factor) promoters. Permits galactose-regulated expression. 
Regulatory gene--GAL4- encodes GAL4 protein, a positive regulator of the 
GALl-MF-alpha 1 hybrid promoter. Derived from S. cerevisiae. 
Terminator--derived from synthetic DNA-based on S. cerevisiae GAPDH 
transcription terminator. 
Signal encoding sequence--Derived from synthetic DNA- based on S. 
cerevisiae PH05 signal. Codons designed substantially for usage preference 
in S. cerevisiae. 
LEU2-d gene--Marker for plasmid selection in S. cerevisiae. Derived from 
plasmid pJDB207. 
2-micron DNA--Derived from plasmid pJDB207. Provides plasmid replication 
origin for S. cerevisiae. 
E. coli replication origin--Derived from pJDB207. 
E. coli selectable marker--Ampicillin resistance--derived from pJDB207. 
Foreign genes are inserted between PH05 signal encoding sequence and the 
GAPDH terminator. 
1. YpGX265GAL4 is digested with restriction endonuclease HindIII and the 
large and small DNA fragments are gel-purified. The small (3.65 Kb) 
fragment is saved for a later step. 
2. The large HindIII fragment is digested with restriction endonuclease 
SmaI, generating two DNA fragments. 
3. M13mp9 (commercially available) is digested with SmaI and HindIII and 
then treated with calf alkaline phosphatase. 
4. The DNA molecules generated in steps 2 and 3 are ligated and used for 
transformation of E. coli. The transformants containing phage 
double-stranded DNA with the yeast promoter-terminator cassette are 
identified. 
5. The recombinant phage double-stranded DNA is digested with restriction 
endonucleases EcoRV and BamHI and the large fragment (approximately 8 kb) 
is gel-purified. 
6. Plasmid pGX2380 (prepared in accordance with Example 7 and containing 
bioadhesive precursor protein cDNA) is digested with restriction 
endonuclease XbaI, and the single-stranded overhang is filled with the 
Klenow fragment of DNA polymerase. 
7. The DNA fragment generated in step 6 is digested with BclI. The small 
DNA fragment (approximately 625 bp) is gel-purified. 
8. Two oligonucleotides of sequence: 
5' ATCAAATCGATGGCGGCC and 5' GGCCGCCATCGATTTGAT are annealed and used in a 
three-way ligation with the DNA fragments generated in steps 5 and 7. 
9. E. coli transformants containing the desired phage DNA molecules are 
identified by restriction endonuclease digestion. 
10. The phage DNA generated in step 9 is digested with restriction 
endonucleases SmaI and HindIII and the small fragment (approximately 1300 
bp) is gel-purified. 
11. YpGX265GAL4 is digested with SmaI and HindIII and the large fragment is 
gel-purified. 
12. The DNA molecules generated in steps 10 and 11 are ligated and E. coli 
is transformed. The desired plasmid, YpGX285, is identified by restriction 
endonuclease digestion. 
13. YpGX285 is linearized with restriction endonuclease HindIII and treated 
with calf alkaline phosphatase. 
14. The 3.65 kb GAL4-containing fragment from step 1 is ligated with the 
linearized YpGX285 generated in step 13. 
15. E. coli is transformed and the desired plasmid, YpGX285GAL4, is 
identified by restriction endonuclease analysis. YpGX285GAL4 was deposited 
on Jan. 29, 1991 at the American Type Culture Collection, 12301 Parklawn 
Drive, Rockville, Md. 20852, U.S.A. in S. cerevisiae host GX9301 and 
assigned number ATCC 74123. 
16. Saccharomyces strains carrying mutations in the LEU2 structural gene 
are transformed with YpGX285GAL4 by standard methods. A suitable strain is 
AH22 (ATCC #38626). 
17. The yeast strain is grown in YNBD medium to maintain the plasmid. The 
YNBD medium contains 0.7% yeast nitrogen base, 2% glucose, and 20 mg/liter 
L-histidine. 
18. For production of the bioadhesive precursor protein the strain is grown 
in medium containing 1% yeast extract, 2% peptone, 1% glucose, and 1% 
galactose at 30.degree. C. with shaking. 
19. Production of the bioadhesive precursor protein is analyzed by Western 
Blot and SDS-polyacrylamide gel electrophoresis of the yeast proteins 
following procedures known in the art. 
EXAMPLE 10 
Growth of GX3015 (pGX2383) and Induction of Bioadhesive Precursor Protein 
Synthesis 
Cultures are maintained using growth temperatures of 30.degree. C. with 
ampicillin at 100 ug/ml and/or medium lacking tryptophan to maintain 
nutritional selection. Plasmid pGX2383 contains the bla gene which encodes 
beta-lactamase providing ampicillin resistance, as well as the trpED that 
in tryptophan-deficient medium complements the trpED102 deletion in the 
GX3015 chromosome. 
A single colony of GX3015 (pGX2383) is picked after growth on minimal salts 
medium (Miller, J. H., "Experiments in Molecular Genetics," Cold Spring 
Harbor Laboratory, 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 (A.sub.600) 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.degree. C. and shaken at 
250 r.p.m. for 6.5 to 9 hours. 
Fermentation is carried out using 8 liters of the following initial medium: 
______________________________________ 
(NH.sub.4).sub.2 SO.sub.4 
30 g 
KH.sub.2 PO.sub.4 15 g 
K.sub.2 HPO.sub.4 5 
Biotin (0.5 mg/ml in 95% ethanol) 
12 ml tap water to 8 liters, 
autoclave 
______________________________________ 
The following additions are made after autoclaving to provide the initial 
medium: 
______________________________________ 
CaCl.sub.2.2H.sub.2 O 
10 ml of 10% (w/v) sterile solution 
glucose 360 ml of 50% (w/v) sterile solution 
niacin 18 ml of 0.5% (w/v) sterile solution 
Trace solution 1 
90 ml 
Trace solution 2 
18 ml 
Trace solution 3 
1.8 ml 
______________________________________ 
The following fermentation conditions are maintained: 
______________________________________ 
pH 7.0 (controlled by 5N NH.sub.4 OH, and 1N H.sub.3 PO.sub.4) 
Sparge rate 1 vvm 
Temperature 32.degree. C. 
Agitation rate 800 r.p.m. 
______________________________________ 
In order to increase cell density prior to induction of expression, a 
system of broth supplementation with nutrients is undertaken. The feed 
solution is prepared as follows: 
1000 g glucose with deionized water for final volume of 1700 mls is 
autoclaved. After autoclaving, there is added: 
______________________________________ 
Trace solution 1 500 ml 
Trace solution 2 100 ml 
Trace solution 3 10 ml 
CaCl.sub.2.2H.sub.2 O 50 ml 
Trace Solution #1 
H.sub.2 O 900 ml 
conc HCl 13.1 ml 
FeCl.sub.3.6H.sub.2 O 5.4 g 
ZnSO.sub.4.7H.sub.2 O 1.44 g 
MnCl.sub.2.4H.sub.2 O 1.0 g 
CuSO.sub.4.5H.sub.2 O 0.25 g 
CoCl.sub.2.6H.sub.2 O 0.24 g 
H.sub.3 BO.sub.3 0.062 g 
Brought to 1000 ml and sterile filtered. 
Trace Solution #2 
H.sub.2 O 900 ml 
HCl 44.8 ml 
MgSO.sub.4.7H.sub.2 O 61.6 g 
Brought to 1000 ml and sterile filtered. 
Trace Solution #3 
H.sub.2 O 1000 ml 
Na.sub.2 MoO.sub.4.2H.sub.2 O 
24.1 g 
Sterile filtered. 
______________________________________ 
The feed solution is initially added to the broth in a volume of 180 ml and 
thereafter as needed to maintain the glucose level at 10 g/liter. Feed 
supplementation is continued until the A.sub.600 reaches 20, at which time 
the cells are induced to express bioadhesive precursor protein from the 
hybrid lambda promoter. Induction is effected by raising the temperature 
to 37.degree. C. to deactivate the temperature-sensitive lambda cI857 
repressor protein produced by the defective lysogen in the GX3015 
chromosome. The fermentation is maintained at 37.degree. C. for another 
6-8 hours. 
EXAMPLE 11 
Purification of Bioadhesive Precursor Protein 
E. coli GX3015 (pGX2383) cells (32 g wet weight) are suspended in 20 ml 20 
mM Tris-HCl, 2mM EDTA (pH 7.5), 1 mM phenylmethylsufonyl fluoride, 25 mM 
iodoacetic acid, and thoroughly disrupted by passage through a French 
press followed by sonication. The cell debris and protein-containing 
inclusion bodies containing bioadhesive precursor protein are pelleted by 
centrifugation at 27,500 g for 30 minutes at 4.degree. 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 supernatant is clear. The 
pellet is then dissolved in 15 ml of 6M guanidine hydrochloride, 5% 
beta-mercaptoethanol, 25 mM iodoacetic acid, and centrifuged at 30,000 xg 
for 30 minutes at 4.degree. C.. The supernatant is dialyzed against 4 
liters of 0.2 mM EDTA, 10 mM iodoacetic acid, with 3 changes which results 
in protein precipitation. The precipitate containing about 0.5 g of 
protein is dissolved in 40 ml of 70% formic acid. Cyanogen bromide (1.3 g) 
is added and the solution is allowed to react overnight at room 
temperature. After rotary evaporation, the residue is extracted with 20 ml 
water (pH 4.0 from residual formic acid). The pH of the water-soluble 
fraction is adjusted to pH 7.0 with 5N KOH resulting in some precipitate 
formation. The supernatant is then applied to a CM cellulose or 
S-Sepharose column (2.5.times.26 cm) equilibrated with 50 mM potassium 
phosphate (pH 7.5). After the column is washed for 14 hours with 50 mM 
potassium phosphate, the bioadhesive precursor protein is eluted with 
either a salt gradient (0 to 0.5M KCl) 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 1). The fractions containing the bioadhesive precursor protein 
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 Sephadex G-75 column chromatography with 0.3M ammonium acetate pH 
4.0. Polyphenolic protein eluting in the first protein peak is dialyzed 
against water and lyophilized for recovering as a salt-free powder. 
The purified protein is hydrolyzed in 6M constant boiling HCl with phenol 
crystal in vacuo at 105.degree. C. for 24 hours. The amino acids in the 
acid hydrolysate are identified as O-phthaldehyde (OPA) derivatives which 
are separated on C.sub.18 reverse-phase HPLC column (Fleury, M. O. and D. 
V. Ashley, Anal. Biochem., 133:330-335 (1983)). The amino acid composition 
is used to verify purity since only a subset of amino acids is present in 
bioadhesive precursor protein. 
EXAMPLE 12 
Alternate Method for Bioadhesive Precursor Protein Purification 
E. coli cells [GX3015 (pGX2383)] or yeast cells [AH22(YpGX285GAL4)] from a 
180 l fermentation are centrifuged in a Westphalia centrifuge, washed with 
saline and resuspended as a 30% solids suspension in 10 mM EDTA, 1.0 mM 
phenylmethylsulfonyl fluoride (PMSF), 10 mM iodoacetic acid (IAA) at pH 
8.0 before breaking by a Manton-Gaulin homogenizer. Bioadhesive precursor 
protein present in an insoluble fraction is collected with the cell debris 
upon Westphalia centrifugation. The centrifugation pellet is made a 20% 
solids suspension in acetic or formic acid at pH 2.2 to 2.5 and mixed for 
several hours to solubilize bioadhesive precursor protein. The extract is 
centrifuged or filtered to remove solids and the clear filtrate solution 
is then adjusted to pH 4 with 5 N KOH in the presence of 10 mM EDTA, 10 mM 
IAA and 1.0 mM PMSF. Impurities are removed as a precipitate by filtration 
or centrifugation. The polyphenolic protein in the clear supernatant is 
then concentrated by ultrafiltration (10,000 M.W. cutoff membrane) and 
subsequently lyophilized. After the residue (approximately 50-100 g) is 
dissolved in 1.2 liter of 70% formic acid, cyanogen bromide (100 g) is 
added and the solution is stirred for 24 hours at room temperature. The 
reaction mixture is then dried by rotary evaporation and the residue is 
dissolved in 50-100 ml 6M guanidine hydrochloride at pH 8.0 and 
centrifuged at 30,000 g for 20 minutes at 4.degree. C. The supernatant is 
chromatographed on a Sephacryl S-300 column equilibrated with the same 
solution. The fractions containing bioadhesive precursor protein are 
collected, adjusted to pH 4.0 with acetic acid, and dialyzed against water 
and recovered as a salt-free powder. 
EXAMPLE 13 
Hydroxylation of Bioadhesive Precursor Protein 
Since tyrosinase has been known to catalyze the hydroxylation of tyrosine 
and oxidation of DOPA (Ito et al., Biochem., 222:407-411 (1984); Marumo 
and Waite, Biochem. Biophys. Acta, 892:98-103 (1986)), mushroom tyrosinase 
or Streptomyces antibioticus tyrosinase can be used to enzymatically 
modify the homogenous E. coli or yeast-produced bioadhesive precursor 
protein. To a 1 ml mixture containing a 2 mg protein, 25 umole ascorbic 
acid and 0.05M sodium phosphate between pH 5 to 7.5, 0.1 mg mushroom 
tyrosinase (Sigma Chemical Co.) is added. The mixture is allowed to react 
at room temperature for 3 hours. The kinetics of the hydroxylation process 
can be monitored by the colorimetric assay for DOPA and DOPA-derived 
quinone (Waite, J. H. and M. L. Tanzer, Anal. Biochem., 111:131-136 
(1981)). The product is further analyzed by amino acid analysis after acid 
hydrolysis as above (Example 9). After correcting for loss during the 
recovery process, amino acid analysis indicates approximately 40% of the 
tyrosine residues are converted to DOPA. 
After hydroxylation, 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 
0.2M acetic acid, or using a membrane filtration method (Amicon PM30, cut 
off of 30,000 M.W.). 
An alternative purification scheme after hydroxylation is described below. 
After CNBr cleavage, the supernatant obtained at pH 7.0 (see Example 11) 
is acidified to between pH 5 and 7 in the presence of ascorbic acid or 
tropolone (Kahn, V. and A. Andrawis, Phytochemistry, 24:905-908 (1985)). 
Hydroxylation is started by the addition of tyrosinase. At the end of the 
reaction, the sample is purified by an SE Sephadex column. The fractions 
containing DOPA are pooled, dialyzed against 2.5% acetic acid and 
lyophilized. The purity of the hydroxylated protein is established by 
acid-urea polyacrylamide gel electrophoresis (Panyium S. and R. Chalkley, 
Arch. Biochem. Biophys., 130:337-346 (1969)) and amino acid analysis. 
The DOPA-containing bioadhesive protein is then ready for formulation as an 
adhesive. 
EXAMPLE 14 
Use of Bioadhesive Protein as a Primer for Conventional Adhesives 
Surfaces such as metals or plastics are frequently given a pretreatment 
such as oxidation with acid, flame treatment or plasma bombardment to 
improve the ability of a surface to "wet" or interact with the adhesive. 
Microbially produced and hydroxylated mussel bioadhesive protein coated 
onto a surface can be used as a pretreatment or priming substance for 
conventional adhesives. An example of the use of mussel bioadhesive 
protein as a primer treatment for bonding two pieces of aluminum is given 
below. 
Hydroxylated bioadhesive protein prepared as in Example 13 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 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. 
As an alternative to speed bioadhesive protein curing and eliminate the 
prehydroxylation step (see Example 13), an enzyme such as mushroom 
tyrosinase (Ito et al., Biochemistry, 222:407-411 (1984)) or Streptomyces 
tyrosinase (Lerch and Ettlinger, Eur. J. Biochem., 31:427-437 (1972)) is 
mixed with the non-hydroxylated protein (Example 11) immediately prior to 
application (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 
quinone species. 
Blends of bioadhesive protein with other polymers (as outlined in adhesive 
examples given below) are also used as primers for other adhesives. 
EXAMPLE 15 
Use of Bioadhesive Protein 
The hydroxylated bioadhesive protein prepared as in Example 13 is dissolved 
at a concentration of 1-700 mg/ml (0.1-70% solids) in water (or a 
physiological salt solution for medical applications) adjusted to pH 6.0 
with dilute acids. 
Immediately prior to application to a surface, 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 concentration 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 o a spray head of a Duploject.RTM. syringe as has 
been described for fibrin sealant (Redl, H. and G. Schlag, Facial Plastic 
Surgery, 2:315-321 (1985)). Alternative enzymes such as mushroom 
tyrosinase (Ito et al., Biochemistry, 222:407-411 (1984)) or Streptomyces 
tyrosinase (Learch and Ettlinger, Eur. J. Biochem., 31:427-437 (1972)) are 
also used. With tyrosinase, the bioadhesive protein need not be previously 
hydroxylated and thus the material without prior hydroxylation described 
in Example 11 may be used. In order for oxidation of DOPA residues to 
quinones to take place and subsequent curing (either with or without 
enzymes), there must be dissolved oxygen present in the bioadhesive 
protein solution. 
EXAMPLE 16 
Composites of Bioadhesive Protein with Other Protein Polymers 
In order to moderate and improve the properties of the mussel polyphenolic 
protein adhesive, blends with other polymers are used. The 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 Enzymol., 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 15 in ratios 
having from 1% to 50% of the solids comprising bioadhesive protein with 
total solids ranging from 10 to 70%. Higher percentages of 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. 
Alternatively, 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. 
In a similar manner, other types of insoluble or crystalline protein sheets 
can be used as reinforcement for adhesive protein. For example, silk 
cloth, or sheets formed from solubilized and reprecipitated alpha-keratose 
from wool keratin fibers (J. De Bersagbes, Curr. Probl. Dermatol., 6:34-86 
(1976)), or polymerized fibrin clot formed from purified fibrinogen, 
thrombin and Factor VIII (Redl, H. and G. Schlag, Facial Plastic Surgery, 
2:315-321 (1985)) are used. For medical applications, the use of sheets of 
fibrin may have the additional benefit of helping to promote wound healing 
(Redl and Schlag, supra). 
EXAMPLE 17 
Composites of Bioadhesive Protein with Carbohydrate Polymers 
Chitosan is dissolved in 1% acetic acid to a concentration of 30-150 mg/ml 
(3-15% w/v). The chitosan solution with a final pH of approximately 6.0 is 
blended with the bioadhesive protein solution described in Example 15. 
Blends typically have bioadhesive protein concentrations between 2 and 30% 
and chitosan concentrations between 1 and 7%. At pH 6.0 where the chitosan 
is still soluble, catechol oxidase and tyrosinase addition catalyzes the 
formation of reactive DOPA-derived quinones and cross-linking. Increasing 
the pH to 8.0 as in Example 15 prior to application results in the 
immediate precipitation of chitosan out of solution which in some cases 
may not be desirable. 
EXAMPLE 18 
Isolation of DNA Clone N1 Encoding M. edulis Bioadhesive Precursor Protein 
Messenger RNA was isolated from fresh M. edulis as described in Example 6. 
Two 18-base probes were synthesized based on the DNA sequence in clone 
14-1. The first synthetic probe (#2214--GTTTGTTGGTTTATATGC) was 
complementary to a conserved sequence close to the 3'-end of the coding 
sequence. This oligonucleotide was also used to prime cDNA synthesis. The 
second probe (#2213-TTTATAAGTTGGCTTTGC) was the complement of the sequence 
that codes for the hexapeptide found in several locations in the cDNA 
clones. Probes 2213 and 2214 were used together since they have the same 
GC/AT ratio and thus approximately the same melting temperature. The use 
of both probes provided multiple hybridization points and a strong signal 
during clone bank screening. 
The clone bank prepared as in Example 6 was screened with the 18-base 
oligonucleotides. Thirty hybridization-positive clones were initially 
identified in duplicate. Twenty-eight of those were plaque purified with 
two rounds of plating and screening, and their DNA was analyzed. A third 
synthetic 18-base probe (#2231-GGGATATATTGACTTGGA), which encodes a 
portion of an uncommon, variant decapeptide, was included in this 
hybridization analysis. As expected, many of the clones had larger cDNA 
inserts than in the cDNA cloning experiments described in Example 4. The 
largest clones, for example N5, N14 and N26, had a primary insert fragment 
of approximately 2.6 kb. Clones N1, N14 and N15 were chosen for initial 
DNA sequence analysis because these DNA preparation were the cleanest. 
Clone N1 had the largest insert that hybridized to all three probes and it 
displayed an intense signal with probe #2213, which encodes the 
predominant hexapeptide sequence. 
An initial sequence analysis of two of the largest clones (N15 and N14) 
showed that both were false positives for polyphenolic protein sequence. 
The 2.1 kb EcoRI insert from clone N1 was sequenced as described in 
Example 6, with the following modifications. The N1 clone could be 
subcloned stably in only one orientation in the M13 sequencing vectors and 
the progressive sequencing strategy of Dale et al., Plasmid, Vol. 13, p. 
31-40, (1985) was performed on only one strand. To confirm that the 
sequence was assembled correctly, the inserts from the series of 
overlapping subclones from the Dale procedure were sized by 
electrophoresis on agarose gels. 
DNA sequencing confirmed that clone N1 contains polyphenolic protein cDNA. 
In fact, this clone appears to encode a very large middle portion of the 
mussel adhesive protein and contains 76 continuous tandem repeats of 
decapeptides and hexapeptides throughout the 2.1 kb length of the clone 
(FIG. 9). Gaps in the sequence shown in FIG. 9 allow optimal alignment of 
the hexapeptides with the decapeptides. Of the 63 decapeptides in the N1 
protein, 31 are identical to the decapeptide described in U.S. Pat. No. 
4,585,585. The variations in each position are shown in FIG. 10. Of the 13 
hexapeptides, 10 are Ala-Lys-Pro-Thr-Tyr-Lys, two have Ser instead of Ala, 
and one has Val instead of Ala. 
Clone N1 does not have a perfect overlap with any of the other cDNA clones 
sequenced to date. While it is possible that N1 lies just upstream of 
carboxy-terminal clones 52 or 14-1, it is also possible that the lack of 
perfect overlap in any of these clones is the result of multiple 
polyphenolic protein genes or alternate splicing patterns, as found in the 
studies of other structural proteins. Clone N1 also does not have a 
characteristic 5'-end sequence and therefore probably does not encode the 
N-terminus. However, it encodes a polyphenolic protein of nearly 80,000 
molecular weight and therefore constitutes the majority of the 
110,000-130,000 intact mussel protein. 
Except for the proline-rich segment of clone 52, these sequencing results 
suggest that the tandem deca- and hexapeptide repeats may constitute the 
complete functional part of this protein with respect to adhesive 
properties. One could easily increase the size of the encoded protein by 
ligating two or more of the sequenced cDNA segments together. 
EXAMPLE 19 
Expression of N1 cDNA in Yeast 
For expression of the N1 cDNA in yeast, it was desirable to position NotI 
and BamHI restriction endonuclease sites at the 5' and 3' ends of the 
coding sequence respectively. To accomplish this, vector M13mp18 was first 
mutagenized by oligonucleotide directed mutagenesis to insert a NotI site 
adjacent to the unique EcoRI site. An oligonucleotide of sequence. 
##STR3## 
was used to prime this mutagenesis. 
The new M13 vector was called MGX463 and has unique NotI and BAMHI 
restriction sites bordering the EcoRI site. MGX463 double-stranded DNA was 
digested with EcoRI and treated with calf-alkaline phosphatase and the 
linear DNA was purified on a 0.8% agarose gel. 
The N1 cDNA coding sequence was removed from an M13mp10 vector by digesting 
double-stranded DNA with EcoRI. The approximately 2200 base pair EcoRI 
fragment was gel purified. This fragment was ligated with the MGX463 
double-stranded DNA which was linearized with EcoRI and treated with calf 
alkaline phosphatase. E. coli strain GX1210 was transformed with the 
ligation mix. This resulted in vector MGX464 which carries the N1 cDNA 
sequence bordered by NotI and BamHI restriction sites. MGX464 was digested 
with NotI and BamHI and the N1 cDNA fragment was gel purified. 
YpGX283 (fully described in commonly assigned co-pending U.S. Pat. 
application Ser. No. 025,243, filed Mar. 12, 1987, and shown in FIG. 11) 
was digested with NotI and BamHI and the large vector fragment was gel 
purified. This fragment was ligated with the NotI/BamHI N1 cDNA fragment 
and E. coli strain GX1210 was transformed, resulting in plasmid YpGX405. 
Plasmid YpGX405 (FIG. 12) is a yeast E. coli shuttle vector which contains 
the N1 cDNA sequence positioned between the PH05 signal coding sequence 
and the GAPDH transcription terminator. Expression is directed by a hybrid 
promoter composed of elements of the yeast GALl and MF-alpha 1 promoters. 
In order to insert the GAL4 gene into YpGX405, that plasmid was linearized 
with HindIII and treated with calf alkaline phosphatase. Vector 
YpGX283GAL4 (fully described in co-pending commonly assigned U.S. Pat. 
application Ser. No. 025,243, filed Mar. 12, 1987), was digested with 
HindIII and the GAL4 gene was gel purified. These two fragments were 
ligated and E. coli strain GX1210 was transformed, resulting in 
YpGX405GAL4. 
To determine if S. cerevisiae produced bioadhesive precursor protein of the 
expected molecular weight, yeast strain D8 transformed with expression 
vector YpGX405GAL4 first grown on YNBD solid medium (0.7% yeast nitrogen 
base, 10% glucose, 2% agar) and then inoculated into 10 ml YPD (1% yeast 
extract, 2% peptone, 2% glucose) so that the initial A.sub.600 reading was 
0.1 and was then grown at 28.degree. C. with shaking for 17-24 hours. The 
cells were harvested and washed with 10 ml YPGal (1% yeast extract, 2% 
peptone, 2% galactose) and resuspended in an equal volume of YPGal and 
induced for 6-28 hours. One ml of that culture was harvested and washed 
with T.sub.25 E.sub.125 pH 8.4 buffer (25 mM Tris-HCl, 125 mM EDTA, pH 
8.4). The cells were then resuspended in 100 ul T.sub.25 E.sub.125 buffer, 
and broken by vortexing in the presence of glass beads. Following the 
addition of 200 ul T25 E125, the cell lysate was removed from the glass 
beads and cell debris was pelleted in a microfuge for five minutes. The 
insoluble pellet was resuspended in 200 ul sample buffer (Laemmli, U.K. 
1970, Nature 227:680-685) and boiled for five minutes. A 25 ul aliquot was 
examined on a 10% SDS-polyacrylamide gel and stained with Coomassie blue. 
The results of this analysis showed that bioadhesive precursor protein of 
the appropriate molecular weight was produced by the yeast strain at 
levels of approximately one percent of the total cell protein. 
EXAMPLE 20 
Purification of Bioadhesive Precursor Protein 
Yeast cells as a 30% suspension in 20 mM Tris pH 7.5, 2 mM EDTA, 0.1 mM 
PMSF, 10 mM iodoacetic acid were thoroughly disrupted mechanically by a 
French Press, sonicator or a Manton Gaulin homogenizer. The adhesive 
precursor protein present in the insoluble fraction was collected with 
cell debris by centrifugation at 25,000 g for 30' at 4.degree. C.. The 
pellet was extensively washed with 10 mM Tris, 1 mM EDTA (pH 7.8) and 
centrifuged as before. The pellet was resuspended as a 30% suspension in 
formic acid (final concentration of 70%) and mixed for several hours. 
Cyanogen bromide (50 gm/1) was added and the suspension was stirred for 24 
hours at room temperature. The reaction mixture was then rotary evaporated 
to dryness, and the residue was resuspended to the original lysis volume 
with water (pH 3.0) and stirred for several hours to extract the adhesive 
protein. 
After centrifugation (as before), the adhesive precursor protein was 
precipitated from the supernatant with 10% NaCl. The precipitate was then 
dissolved in guanidinium chloride (final concentration 6M, pH 8.0) with 5% 
2-mercaptoethanol and chromatographed on a Sephacryl S-300 or S-400 
column, depending on the size of the adhesive precursor protein. The 
fractions containing the adhesive precursor protein were pooled, adjusted 
to pH 4.0 with glacial acetic acid, dialyzed against 5% acetic acid, then 
0.1% acetic acid, with several changes of the dialysis solution, and 
lyophilized. 
The purified protein was characterized by its mobility on 
SDS-polyacrylamide gel electrophoresis using both Coumassie blue stain and 
Western blot analysis, its UV absorption spectrum, protein quantitation 
and amino acid composition analysis. 
EXAMPLE 21 
The yeast cells were processed as in Example 20 up to the 10% NaCl 
precipitation step. Instead of treating with NaCl, the supernatant was 
adjusted to pH 5.2 and centrifuged. The clear supernatant was dialyzed 
against 10% formic acid and slowly to water and then to 30 mM phosphate 
buffer (pH 7.3). After centrifugation, the supernatant was chromatographed 
on a weak cation exchanger. The column was eluted first with a 0.2-1M salt 
gradient at pH 7.3, followed by 1M NaCl in 5% acetic acid. The fractions 
containing the adhesive precursor protein were pooled, dialyzed and 
lyophilized as in Example 20, and the identity and purity of the adhesive 
precursor protein were determined as described in Example 20. 
EXAMPLE 22 
After the yeast cells were broken and centrifuged as in Example 20, the 
pellet was solubilized in either 6M guanidinium chloride or 8M urea with 
5% 2-mercaptoethenol. The insoluble cellular components were removed by 
centrifuging at 25,000 g for 30' at 4.degree. C. The protein in the 
supernatant was precipitated by dialysis against water, and the adhesive 
precursor protein was extracted by a dilute formic acid or acetic acid 
solution (pH 2.0) for several hours. The adhesive precursor protein was 
precipitated by either adjusting the pH to neutral, or making the solution 
10% NaCl. After centrifugation, the adhesive precursor protein pellet was 
dissolved in 6M guanidinium chloride and chromatographed on an S-300 or 
S-400 column depending on the size of the protein. The adhesive precursor 
protein, with the correct size, was pooled, adjusted to pH 4.0 with acetic 
acid, dialyzed against 5% acetic acid then 0.1% acetic acid and 
lyophilized. 
EXAMPLE 23 
The yeast cells were broken and thoroughly washed as in Example 20. The 
pellet was extracted for several hours with dilute formic or acetic acid 
(pH 2.0). The solution was then adjusted to pH 3.0 and centrifuged. The 
adhesive precursor protein present in the supernatant was precipitated 
when the pH was adjusted to between 5.0 and 5.5. The adhesive precursor 
protein was collected by centrifugation, and, if necessary, further 
purified by either a weak cation exchange, or a gel filtration column as 
described in Examples 20 and 21.