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Figure 1. The adhesive protein synthesis pathway in a typical marine invertebrate, the mussel, and the molecular tools used to characterize protein‐based adhesives. The genes encoding adhesive proteins are transcribed into mRNAs, which are then translated into protein precursors. These, in turn, can be post‐translationally modified to give mature adhesive proteins. Nucleic acids (DNA and mRNA) can be extracted from the adhesive organ(s) (here, the mussel's foot). Both can be submitted to next‐generation sequencing to obtain, respectively, the genome of the animal or the transcriptome of the adhesive organ(s). Proteins, on the other hand, can be extracted from the secreted material (here, the byssus) or the adhesive organ(s) and submitted to peptide sequencing by tandem mass spectrometry (MS/MS). The obtained peptide sequences can be used for a basic local alignment search tool (BLAST) search in the genome or transcriptome, allowing the recovery of the full‐length sequence (if available) of the cDNA coding for the investigated protein. |
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Figure 2. Scientific publications reporting the production of marine adhesive proteins. (A) With increasingly more adhesive proteins being described in various marine invertebrates and additional biomedical applications being sought, the number of articles comprising recombinant protein production has risen steadily in the last 20 years. (B) In this review, we catalogued around 120 publications. Up to the end of 2024, 6 phyla of marine invertebrates have inspired the production of recombinant adhesive proteins, albeit with varying degrees of effort, as reflected by the number of publications. The phylogenetic relationships between these organisms are shown on the left (based on).[
2
]
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Figure 3. General outline of major steps for recombinant protein production and purification. First, the sequence coding for the protein of interest is obtained either by PCR amplification from mRNA extracted from the adhesive organ or through gene synthesis by a specialized company. The latter approach allows for codon optimization, overcoming limitations associated with inter‐species differences in codon usage, thereby enhancing protein production yield. The sequence is then cloned into an expression vector, in frame with a sequence coding for a tag (to facilitate protein purification and detection or to improve protein expression and solubility), or for another protein (e.g., to bring new functionalities). After transferring the vector into host cells (transformation/transfection), protein expression is induced. Proteins can sometimes be recovered from the culture medium, however, most of the time, the cells are collected and lysed to recover the proteins. The proteins can be found in the soluble fraction or as insoluble aggregates, known as inclusion bodies. In the latter case, they need to be extracted with denaturing buffers. Solubilized proteins are then purified using one or several purification methods to obtain pure proteins. Finally, the proteins are concentrated and refolded (usually through progressive buffer exchange), and may undergo additional processes, such as enzymatic treatment, to restore native function. |
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Figure 4. Marine invertebrate adhesive systems that were used as models for the production of recombinant adhesive proteins. (A) Example of an intertidal community illustrating the six metazoan taxa in which adult individuals rely on different types of adhesion to attach to the substrate: the permanent adhesion of bivalves, barnacles, tubeworms and ascidians; the transitory adhesion of sea anemones; and the temporary adhesion of sea stars (modified from[
2
]). (B) In two of these groups, the larval stages attaching to the substratum during settlement and metamorphosis (the ascidian tadpole larva and the barnacle cyprid larva) also inspired the production of recombinant adhesive proteins. Insets in colored circles (same color code as in Figure 2) show details of the adhesive organs with the adhesive secretions highlighted in red. In the case of tubeworms, the inset shows the structure of the tube consisting of sand grains glued together by cement spots. |
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Figure 5. Mussels attach to surfaces using an acellular extracorporeal structure known as the byssus. (A) Each byssal thread is formed by proteins produced by four glands within the foot (modified from[
22
]). The stem gland produces the attachment stem (dark blue) which originates in the soft tissue; the core gland provides the proteins within the thread core (light blue); the plaque gland provides the bulk and interfacial proteins (orange) for attachment to any underwater substrate (Su); and the cuticle gland produces the cuticle covering both the thread and the plaque (purple). (B) Proteins constituting the byssus (only proteins whose sequence has been used to produce recombinant proteins are displayed. Prepepsinized Collagens (PreCol's), thread matrix proteins (TMPs) and proximal thread matrix proteins (PTMPs) are made by the core gland and found within the core. The 3 PreCol proteins consist of 3 different domains flanking an atypical collagen domain, while PTMP contains von Willebrand Factor A domains. Mussel foot protein‐1 (mfp‐1) contains many tandem repeats and provides the protective coating. Mfp‐2‐6 are examples of the proteins found within the adhesive plaque. Notably, only mfp‐2 is distinguished with domains; it contains multiple EGF‐like domains which form the porous bulk of the adhesive plaque. (C) Most mussel byssal proteins have been shown to contain DOPA, which is formed by post‐translational conversion of tyrosine resides by tyrosinase. The catechol group of DOPA residues can contribute to both adhesive adsorption on the substrate and byssus cohesion. |
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Figure 6. The adhesive proteins of scallops, adult tunicates, and sea anemones that have been produced recombinantly. In these organisms, the specific glands or secretory cells producing these proteins have not been identified. (A) Three scallop byssus proteins have been identified and recombinantly produced: sbp5‐2, sbp8‐1, and sbp9. Although sbp8 has been synthesized full‐length, only fragments of sbp5‐2 and sbp9 have been produced. (B) The sea squirt protein ASP‐1, characterized by one von Willebrand type A domain, has been produced recombinantly in insect cells. (C) In sea anemones, only one adhesive protein, TSRL, which is comprised of three repeats of thrombospondin type 1, has been produced recombinantly. |
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Figure 7. The adhesive system of adult barnacles. (A) Section through an acorn barnacle showing the organization of the cement apparatus (red) which consists of clusters of cement cells located in the basal part of the organism (modified from[
147
]). After synthesis, cement proteins are delivered to the interface between the barnacle's base and the substrate (Su) through a series of ducts. (B) Among the different proteins constituting the adult cement, six have been produced recombinantly. Most cement proteins are made up of combinations of hydrophilic, hydrophobic, or charged regions except for SICP which is an alpha‐2‐macroglobulin‐like protein. |
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Figure 8. Schematic drawing of the adhesive system in tubeworms of the family Sabellariidae. Different adhesive proteins are packaged in one of two types of cement cells (containing either homogeneous granules or heterogeneous granules) located in three anterior segments of the worm. Granules from both types of cement cells travel through long cell processes and are secreted by the building organ, a structure located near the mouth. Once secreted, their contents coalesce to form the porous adhesive spots cementing sand grains together to build the tube in which the worm is living. To date, only one adhesive protein, Sa‐1, has been produced recombinantly. It is produced by cement cells with heterogeneous granules and comprises seven glycine‐ and tyrosine‐rich repeats. |
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Figure 9. The duo‐gland adhesive system of sea stars. (A) At the tip of sea star tube feet, the epidermis encloses three types of secretory cells ‐i.e., type 1 and 2 adhesive cells (AC1 and AC2) and de‐adhesive cells (DAC)‐ interspersed with support cells (SC) (modified from[
162
]). When a tube foot attaches, type 2 adhesive cells release their contents, surface active proteins, which form a homogeneous film covering the substrate (Su). Concomitantly, type 1 adhesive cells release proteins with a bulk function, which form a thick cohesive meshwork structure. (B) Sfp1 is a large multimodular adhesive protein produced by type 1 adhesive cells. Thanks to its different functional domains, it can presumably interact with multiple partners and makes up the structural scaffold of the adhesive footprint that remains on the substrate after tube foot detachment. Two fragments of sfp1 have been produced recombinantly; the C‐terminal part of the β subunit and the δ subunit. |
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Figure 10. Hybrid protein design and their improved performances. (A) The DNA sequence coding for an adhesive protein can be cloned into an expression vector in frame with a sequence coding for another protein. After production, the resulting hybrid protein usually displays new functionalities. (B) The hybrid protein fp‐151, obtained by the fusion of decapeptides from the mussel cuticle protein mfp1 at each terminus of the interfacial adhesive protein mfp5, increased expression yield three‐fold compared to recombinant mfp5 alone (data from[
221
]). (C) Coatings made up of the hybrid protein Sbp9Δ‐LL37, obtained by the fusion of the partial scallop adhesive protein Sbp9Δ with antimicrobial peptide LL37, displayed antimicrobial activity whereas coatings consisting of Sbp9Δ alone did not (data from[
222
]). (D) The hybrid protein CsgA‐cp19 k, obtained by the fusion of bacterial curli protein CsgA with barnacle cement protein cp1 9k, demonstrated improved self‐assembly into amyloid fibrils compared to recombinant cp19k alone as evidenced by Thioflavin T fluorescence after a 16 h incubation in artificial seawater (data from[
223
]). |
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Figure 11. Examples of biomaterials fabricated through the association of mussel adhesive proteins (MAPs) with other components such as polysaccharides, ions, or synthetic materials. The hydrogels, fibers, or films assembled in this way were further processed or combined to fabricate complex biomaterials for various medical applications. (A) A bone graft binder has been obtained through the complex coacervation of negatively charged hyaluronic acid (HA) with a positively charged recombinant MAP. The rMAP/HA coacervate stabilized the agglomerated deproteinized bovine bone minerals (DBBM)and promoted in vivo bone regeneration. Reproduced with permission.[
255
] 2016, Wiley. (B) Nanoparticles (NP) based on Fe3+–DOPA complexation with recombinant DOPA‐containing MAP were synthesized using an electrospraying process and loaded with doxorubicin (DOX). In vitro, the DOX‐loaded NPs released the associated drug by changing the pH. Fluorescence microscopy images of HeLa cells incubated with DOX‐loaded NPs for one and three hours showed the cellular uptake behavior of DOX. Reproduced with permission.[
256
] 2015, Wiley. (C) Customized underwater bioadhesive patches (CUBAPs) have been fabricated by ultraviolet (UV) crosslinking of MAP‐(meth)acryloyl with (meth)acrylic acid to form poly((meta)acrylic acid). CUBAPs display switchable underwater adhesiveness: initially dry and nonadhesive, they absorb moisture when applied to living tissues and develop strong surface adhesion based on different molecular interactions. CUBAPs have been evaluated ex vivo and in vivo for healing wounds in diverse internal organs and implanted electronic devices. Reproduced with permission.[
257
] 2024, Wiley. (D) Drug‐loaded magnetic microparticles have been prepared by incorporating iron oxide (IO) magnetic nanoparticles and DOX in a MAP matrix (MAP@IO MPs). The bioengineered MAPs were cross‐linked through their lysine residues by using genipin. Esophageal cancer‐mimicking microchannels were used to evaluate the magnetic capture of the MAP@IO (red fluorescence). The high capture efficiency of this material led to decreased cancer cell viability. Reproduced with permission.[
258
] 2021, Wiley. |
