Random mutagenesis of amelogenin for engineering protein nanoparticles

Abstract

Nanoparticles made from recombinant proteins offer excellent potential for several nanotechnological applications. However, only a very limited number of proteins are currently being used for such purposes due to limited availability and stability. Therefore, we have investigated the enamel matrix protein amelogenin as a new alternative protein for use as recombinant nanoparticles. Amelogenin is a robust protein that has the ability to self-assemble into nanosized particles termed nanospheres. This self-assembly property of amelogenin is highly pH-dependent, and modifications of the solubility behavior for amelogenin can be particularly important for some applications such as drug delivery, where responsiveness at a specific pH is an essential property. In this study, an amelogenin mutant library was created and used to screen amelogenin variants with modified solubility/aggregation profiles in response to externally applied pH changes. Fifty amelogenin mutants were identified and produced recombinantly, purified and characterized. Several mutants with distinct solubility profiles were obtained that could form uniform nanospheres, ranging from 30 to 60 nm in hydrodynamic diameter. The mutants displayed a shifted onset of pH-dependent aggregation compared to wild-type amelogenin. At physiological pH, some mutants formed soluble nanospheres, while others generated nanosphere aggregates, suggesting different practical uses for the different mutants. By mixing and co-assembling mutant and wild-type amelogenin at different ratios, the level of nanosphere aggregation could be tuned at a given pH. By exploring combinations of different amelogenin variants it is possible to control aggregation events in nanomedical applications where a specific pH response is required.

Functionalization of Recombinant Amelogenin Nanospheres Allows Their Binding to Cellulose Materials

“…C. The pH solubility profile of rh174 exhibited a U‐shape with the lowest solubility close to pH 6.5, as in previous reports . A similar profile was observed for rh174CDB, but the fusion protein displayed a low solubility over a wider pH range.…”

“…This has also recently been shown by others . Besides amelogenin fusion proteins, we have also reported that a random mutation amelogenin library can be used to create amelogenins with distinct solubility behavior, further demonstrating the possibilities to engineer the properties of this protein.…”

“…). Previous studies have shown that different amelogenin variants can be mixed at non‐aggregating conditions and co‐assembled into composite amelogenin aggregates with distinct properties that depend on the proportion of the individual amelogenin variants . In the present study we observed a difference in solubility when rh174CBD and rh174 were mixed (Fig.…”

“…The ability of amelogenin to self‐assemble into nanosized aggregates makes it an important protein to explore as a building block for nanomaterials . Previously we have described a method for producing recombinant amelogenin that enables simple production of large quantities of the protein .…”

Amyloid-like ribbons of amelogenins in enamel mineralization

“…It has been previously shown that the C-terminus has been associated with HAP binding. In an attempt to induce mineralization of amelogenin nanoribbons, we modified its C-terminus by extending it with a triple repeat of amelogenin sequence KTKR 47 48 , and tested the effect of additional electrostatic charges on self-assembly and mineralization. The resulting rH174-(+9) protein was exposed to the same self-assembly protocol as described above and also assembled into ribbons that were organized in a parallel fashion, but with extremely regular distances of about 69 nm between ribbons when immobilized on glass surfaces ( Fig.…”