Development of an universal affinity fusion tag (Poly‐DOPA) for immobilizing recombinant proteins on biomaterials

Abstract

The covalent and non-covalent immobilization of growth factors such as recombinant human bone morphogenetic protein 2 (rhBMP-2) on metals and bone replacement materials in bioactive form is a recent development. Up to now the immobilization technology usually involved the chemical modification and activation of the biomaterial surface followed by attachment of the bioactive protein.Here we suggest an alternative method in which an affinity tag fused to an active protein will allow immobilization without additional chemistry. For biomaterials such as minerals, metals (titanium, steel, CoCrMo), glass ceramics, teflon and possibly bone and teeth ideal adhesion molecules would be the foot proteins (Mefps) of the mussel M. edulis which contain the rare amino acid dihydroxy phenylalanine (DOPA). Recently it could be shown by Messersmith's group that a single DOPA-molecule can be noncovalently bound to titanium dioxide surface with a dissociation energy of 22.2 kcal/mol (Lee, H.; Scherer, N. F.; Messersmith, P. B. Proc. Natl. Acad. Sci. U. S. A 2006, 103, 12999 -13003

Tyrosinase-catalyzed site-specific immobilization of engineered C-phycocyanin to surface

“…In line with previous studies showing the use of tyrosine-containing tags for enzymatic protein crosslinking2729303132, we proved that tyrosines added to a protein within an unstructured region, e.g. a peptide, are not only specific sites of crosslinking but can also be used for site-specific protein immobilization.…”

“…This work shows that the production of enzyme-catalyzed covalently crosslinked, fluorescent, high-molecular weight aggregates of a fluorescent protein with possible application in the biomedical and diagnostic field is possible using a crosslinking enzyme such as tyrosinase instead of chemical crosslinkers. In line with previous studies showing the use of tyrosine-containing tags for enzymatic protein crosslinking 27 29 30 31 32 , we proved that tyrosines added to a protein within an unstructured region, e.g. a peptide, are not only specific sites of crosslinking but can also be used for site-specific protein immobilization.…”

Enzymatic mineralization of gellan gum hydrogel for bone tissue-engineering applications and its enhancement by polydopamine

“…The effectiveness of PDA functionalization in promoting mineral formation was demonstrated by increases in solid mass fraction (Figure ), mass percentage attributable to mineral (Figure ) and stiffness (Figure ). The effect of PDA was particularly remarkable, considering that ALP could have been released during the incubation in DA solution, which lasted up to 24 h, although PDA formation in the gel could have hindered ALP release since PDA has shown affinity for proteins (Jennissen and Laub, ).…”

Facile Plasma‐Enhanced Deposition of Ultrathin Crosslinked Amino Acid Films for Conformal Biometallization

“…Bioenabled methods are used to form inorganic coatings and nanoparticles composed of, for example, gold, silver, platinum, titania, silicates, calcinates, and iron, through the reduction of the corresponding ions with biomolecules (DNA, amino acids, proteins, and peptides), which act as nucleation sites, templates, and reducing agents 1–10. A wide range of applications makes the method extremely useful for directed assembly, in which a targeted surface can be coated with the biomolecules and exposed to a solution containing the desired ions to be deposited.…”