Adhesive proteins are industrially important proteins which were isolated and from the mussels and they were produced using conventional methods such as natural extraction. But now this protein is produced using genetic engineering technologies. It has wide applications in various arena.

1. ADHESIVE PROTEINS
Presentation by : Nehali N Buchade
MES Abasaheb Garware College of arts,
science and commerce, Pune.

2. What is a mussel?
• Mussel is the common name for members of several families of bivalve molluscs,
from saltwater and freshwater habitats. These groups have in common a shell whose
outline is elongated and asymmetrical compared with other edible clams, which are
often more or less rounded or oval.
• In nature, mussel adhesive proteins (MAPs) show remarkable adhesive properties,
biocompatibility, and biodegradability.
• Humans have used mussels as food for thousands of years.
• About 17 species are edible, of which the most commonly eaten are Mytilus edulis,
M. galloprovincialis, M. trossellus and Perna canaliculus.
• Sandcastle worms, and endoparasitic worms can also be used to produce the adhesive
proteins.

3. What are adhesive proteins?
Marine mussels secrete the protein
based- adhesives, which enable them
to anchor to various surface in a
saline, intertidal zone.
These are secreted in the liquid form which
then solidify to form a byssal thread.
Atleast 6 Mfps have been identified from the adhesive
plaques of several species of mussel (Mfp 1 to Mfp6).

4. • Mussel foot proteins contain a large amount of catecholic amino acid, DOPA in their
protein sequences.
• Catechol helps in robust and durable adhesion to various substrate surfaces .
• This property highlighted the need for developing synthetic mussel-inspired adhesive
polymers with strong water resistant adhesive properties.

5. Applications of adhesive proteins:
Hard
tissue
adhesives
Byssal threads Environmental
aplications
Tooth
cement
Soft tissue
applications

6. Applications of adhesive proteins:
1. Hard tissue adhesives
Bone and tooth cements are the most used examples of hard tissue adhesives.
In joint replacement surgery, the bone cement fills and localizes in the space
between the implant and the bone, thereby acting as an implant fixation and
transferring the mechanical load from the implant to the bone.
2. Tooth cement
They help in sealing or fixing and casting the filling substance to both the dentin
and enamel.
3. Environmental applications
•Mussels are widely used as bio-indicators to monitor the health of aquatic
environments in both fresh water and the marine environments.
•They are particularly useful since they are distributed worldwide and they are
sessile.
•These characteristics ensure that they are representative of the environment where
they are sampled or placed..

7. Their population status or structure, physiology, behavior or the level of
contamination with elements or compounds can indicate the status of the
ecosystem.
4. Byssal threads – these are used to anchor mussels to substrates, are now
recognized as superior bonding agents. A number of studies have
investigated mussel "glues" for industrial and surgical applications.
Additionally byssal threads have provided insight into the construction of
artificial tendons.
5. Soft tissue adhesives:
Bioglues or sealants and patches. Bioglues are usually applied as surgical
adhesives in cardiovascular, neurological, and soft tissue surgeries.
One such example, BioGlue (Levi BioTECH), was demonstrated to lessen
bleeding during cardiac procedures (e.g., aortic dissection, and replacement and
implantation of biomedical devices).

9. • Marine-derived protein bio-adhesives are considered promising biomaterials
•To live in tidal aqueous environments and to protect themselves from predators,
marine sessile organisms, such as mussels and tubeworms, attach themselves to hard
substratum using protein-based adhesives.

10. Fig: Adhesion mechanism of marine mussel foot proteins (Mfps). A. representaion
sequence of Mfp-3 from mussel plaque. B the amino acid sequence of the Mfp-3 .
C mussel adhesion with the catechol moiety. E. Ultrastructure of a byssal adhesive
plaque.

12. Natural extraction
This method was initially used to isolate mussel adhesive protein for commercial
purposes, but it was labour intensive and inefficient, it used to require 10,000 mussels for 1
gm of protein.
Successful mass production technologies have not yet been developed for mussel
adhesive proteins.
Mussel foot protein type 1 (fp-1) is considered as a key protein for adhesion of mussels
in wet environments, it contained ~ 13 mol% DOPA and consisted of about 80 repeats of a
decapeptide consensus sequence.
 The expression of this protein has been tried in several expression systems such as yeasts
and E. coli.
 However these attempts failed to express functional and economical MAP’s for several
reasons.
 It had a highly biased amino acid composition (5 a.a types comprised of 89% of the
total amino acids), the need of a huge number of tRNA , and small amount of adhesive
produced.

13. SYNTHESIS OF ANIMALADHESIVE USING MICROBIAL CELLS:
1.How it was synthesized?
Researchers were trying to produce an animal adhesive biopolymer using the
blue mussel – Mytilus edulis, mytilus edulis foot protein 5 (mefp-5) in microbial
cells.
This biopolymer is very strong, waterproof, adhesive protein , called byssal
adhesive. This adhesive helps the mussel to attach very tightly to a variety of
surfaces.
2.Problems associated with the isolation of byssal adhesive :
This adhesive protein becomes highly cross-linked and it was difficult to
sequence.

14. 3. Then how this protein was isolated??
• They tried to isolate an intracellular precursor form of the adhesive protein,
called the 130- kDa precursor protein, that could be analyzed biochemically.
• It was found that the precursor protein is rich in serine, threonine, lysine,
proline, and tyrosine : 60-70% of the amino acids contained a hydroxyl group
Most of the proline residues are hydroxylated to 3,4 hydroxyproline (Hyp), and
the majority of the tyrosines are hydroxylated to 3,4 dihydroxyphenylalanine
(DOPA).
High levels of Dopa content are observed in mussel adhesive proteins (MAPs;
3~30 mol%) and tubeworm cement proteins (7~10 mol%).

15. Amino acid sequence analysis of byssal protein was done:
•It was revealed that the byssal protein is composed of large repeating units that
consists of decapeptide with the sequence- Ala-Lys- (Pro or Hyp)-Ser-( Tyr or
DOPA)-Hyp-Hyp-Thr-DOPA-Lys; 7 of these 10 amino acids are hydroxylated.
•The cDNA for the 130- kDa precursor adhesive protein was isolated from a
cDNA library .
•The gland of the mussel –Mytilus edulis secretes the byssal adhesive. The
messenger RNA was isolated from this gland and the cDNA library was
constructed.

16. The problems during cloning, expression and production of the adhesive
proteins:
•The features of the adhesive protein and the cDNA were unusual which made the
cloning, expression and production in a heterologous host of a functional adhesive protein
difficult.
•Proline, lysine and tyrosine represent about 70% of the amino acids of the protein.
•Therefore very high levels of synthesis was not achievable because the corresponding
intracellular aminoacyl- t RNA pools might be limiting.

17. How the problems were deduced??
When either complete or partial cDNAs for the adhesive protein were cloned onto
yeast expression vectors and introduced into yeast cells, active form of the adhesive
proteins were synthesized ranging from 20-100 kDa and it represented about 2-5%
of the total cell protein.
Thus, there were no problems with the cloned c DNA or the production of
moderate amounts of the adhesive proteins.
Higher expression levels were attained when a chemically synthesized adhesive
protein gene sequence was used.

18. How was the protein produced using E.coli??
 Repeating DNA units that encode the consensus decapeptide repeat of the adhesive
protein were used to construct a 600 base pairs synthetic gene that encoded a protein with
a mol mass of approx 25 kDa.
 The 30 bp repeat, was used as a fundamental building block of the synthetic gene, this
gene consisted of a codon that were optimized for E.coli.
 T7 was used as the promoter for the expression of this synthetic gene at very high
levels.

19. Fig: T7 promoter sequence.
•Microorganisms limit in their ability to hydroxylate amino acids
posttranslationaly, so the final protein that they made was unhydroxylated.
•A number of tyrosine residues of the protein were not converted to DOPA,
which had a disadvantage /inability of the proteins to form cross-links.

20. How this deficiency was overcome??
• This deficiency was overcome by creating an in vitro hydroxylation system that
made use of a bacterial tyrosinase in the presence of ascorbic acid to hydroxylate the
tyrosine residues to DOPA.
• Ascorbic acid was included in the reaction mixture to prevent the premature
oxidation of the DOPA residues to o-quinone.
What was the need of controlling the oxidation??
• Oxidation must be controlled because it leads to cross- linking of the adhesive
protein subunits.
• The cross-linking must not occur prior to its use.
• When the precursor form of the adhesive protein is oxidized, the cross linked protein
can bind to a variety of surfaces such as polystyrene, glass, hydrogel and collagen.

21. •The protein can be strengthened by adding other proteins to the adhesive protein mixture
before oxidation and cross-linking.
•The addition of the amounts of the accessory proteins, adhesives that have unique
properties can be created.
Fig: Pathway for in- vitro posttranslational hydroxylation of some of the tyrosine
residues in the M. edulis adhesive protein. Tyrosine is converted to DOPA by the action
of the enzyme tyrosinase and then can be oxidized to o- quinone by either catechol
oxidase or tyrosinase. The oxidation of the DOPA to 0- quinone can be prevented by
the addition of ascorbic acid.

22. Co-expression system to obtain in vivo modified MAP:
• Additional in vitro tyrosinase modification was not needed to obtain adhesive
properties and the in vivo modified MAP showed superior adhesive strength compared
to in vitro modified protein.
• Successfully over-expressed fp-151 (~40% of total protein) using a pET vector
system with strong T7 promoter and pBR322 origin (right vector map).
• Because the pET vector can be used in combination with a vector system with p15A
replicon and functional expression of tyrosinase from Streptomyces antibioticus was
reported in E. coli under the control of T7 promoter, recombinant pACYC-Tyr-438
plasmid (left vector map) was prepared from pACYCDuet-1, which carries the p15A
origin.

23. Fig: Co-expression of fp-151 and tyrosinase. In- vivo method
Two vectors – were used in this method to produce the tyrosine and the mussel
adhesive protein simultaneously.

24.  For functional expression of recombinant tyrosinase, ORF 438, which is essential for
copper incorporation into the active site of tyrosinase, was also co-expressed under the
T7 promoter.
 Then, both recombinant pET-fp151 and pACYC-Tyr-438 plasmids were introduced into E.
coli BL21(DE3) to obtain in vivo tyrosine-modified fp-151.
 Recombinant fp-151 was successfully co-expressed with tyrosinase and ORF 438 in the
dual vector system . It was found that its expression level was similar to that of fp-151 from
pET-fp151 alone.
 It is expected that this co-expression strategy will accelerate the use of functional MAPs in
practical applications and can be successfully applied to prepare other Dopa/Dopaquinone-
based biomaterials.

25. REFERENCES:
Joseph P. Park, a Min-Jung Choi, b Se Hun Kim, c Seung Hwan Lee, b Haeshin Lee January 2014
Volume 80 Number 1 Applied and Environmental Microbiology p. 43–53
Preparation of Sticky Escherichia coli through Surface Display of an Adhesive Catecholamine
Moiety Received 8 July 2013 Accepted 6 October 2013 Published ahead of print 11 October 2013
https://en.wikipedia.org/wiki/Mussel
file:///C:/Users/NISHA/Downloads/s40824-017-0101-y.pdf
Park et al. Biomaterials Research (2017) 21:16 DOI 10.11
Advances in medical adhesives inspired by aquatic organisms’ adhesion Kyu Ha Park, Keum-Yong
Seong, Seung Yun Yang and Sungbaek Seo.
Pegah kord Forooshani, Bruce P. lee et al Journal of polymer science Part A: Polymer chemistry/
volume 55, issue 1 recent approaches in designing bioadhesive materials inspired by mussel adhesive
protein.