Molecular Diversity And Recombinant Expression Analysis Of Mytilus Coruscus Foot Protein

Mussels attach to solid surfaces in the sea using a holdfast named byssul, whichmakes mussel adhesion strong and tough to prevent mussel from dislodged and sweptaway by waves. The byssus of Mytilus coruscus resembles a bundle of threads and consistsof two parts: thread and a flared adhesive plaque, which is distally tipped at the thread endsand directly attached to foreign surfaces. The byssus of marine mussels, containing a suitof proteins that are peculiar in having high levels of dopa, has recently provided importantchemical and physical insights into moisture-resistant adhesion. To date, about elevendeferent proteins have been described from mussel byssus, including six mussel footproteins (mfp-1~6), three prepolymerized collagens (preCOLs), thread matrix proteins (i.e.,tmp-1), and polyphenol oxidase with no homogenous purification.In the mussel foot proteins, The amino acid3,4-l-dihydroxyphenylalanine (DOPA)plays key role in mussel adhesion, thus, the definitive detection and quantification ofDOPA in proteins are essential. Using nitroblue tetrazolium/Glycinate (NBT/Glycinate)specific staining, quantitative analysis of DOPA was performed with Mytilus coruscusbyssal proteins, together with electrophoresis and coating assay, the DOPA content, proteindistribution, and adhesive ability of foot proteins from thread and plaque were compared.Using these strategies, we found that the average DOPA content in proteins of plaques isabout2.33times higher than that of thread; adhesive proteins of byssal plaque have higherDOPA content and lower molecular weight distribution than that of byssal thread; moreover, the coating ability of proteins from plaque on glass was greater than that from threadat the same protein concentration; PDQuest analysis showed that the intensity of spotsfrom plaque is about four-fold more than that from thread. The difference of DOPAcontent in byssal thread and plaque is in accordance with the different protein compositionof the two parts of byssus, which reveals the functional diversity of adhesive proteins inmussel adhesion.Mussel foot proteins identified so far have been suggested as a promising source of awater-resistant bioadhesive for potential use in biotechnological applications and medicaladhesives. While, The protein composition of mussel byssus have not yet been fullydefined for the reasons of the relatively low abundance and water-insoluble of musselbyssal proteins. In this study, we developed one of the first proteomic approaches appliedto byssus from Mytilus coruscus for further understanding the molecular composition and screening novel foot proteins from mussel byssus. Foot proteins extracted by aceticacid-urea and acetic acid-guanidine chloride were separated by Two-dimensional gelelectrophoresis (2-DE) and analyzed by MALDI-TOF/TOF. MS/MS spectra were searchedagainst the EST sequences available in the public database for Mytilus. Using thisapproach, more than35foot proteins were detected, and we were able to identify partial orfull-length sequence transcripts that encode for novel byssal proteins, including three novelfoot proteins whose sequences do not present any homologous proteins or alreadydescribed foot proteins’ domains.In priviously studies, mfp-3,mfp-5and mfp-6had been suggested as the mainadhesive protein in the plaques closest to the adhesion interface and been the focus ofsubstantial biomaterials development research within the last decade. The cDNA gene ofthese mfp were cloned and transferred to E. coli and Pichia stipitis, respectively. Therecombinant mcofp3fused with a hexa-histidine affinity ligand was successfully expressedthrough an Escherichia coli expression system, and the recombinant mcofp3was purifiedusing affinity chromatography followed by reverse phase high performance liquidchromatography (HPLC). The DOPA content and adhesive properties of purifiedrecombinant mcofp3with or without tyrosinase modification were compared with thenative mcofp3. These assays showed that recombinant mcofp3has significant adhesiveability and may be useful as a bioadhesive in medical or underwater environments. Whilethe research of recombinant expression of mfp-5and mfp-6are undergoing.