Recombinant amelogenin - Strategies for purification and protein processing

Sammanfattning: Amelogenin is an extracellular matrix protein involved in formation of enamel during early tooth development. By interacting with the forming enamel crystallites amelogenin affects their growth, so that highly elongated hydroxyapatite crystals are formed. Early during the enamel formation amelogenin is proteolytically processed, which is essential for correct enamel formation. However, the exact mechanism of amelogenin in enamel formation is not fully understood. Amelogenin has also been found to have signal molecule-like properties, which suggests that amelogenin has other functions during tooth development. Expression of amelogenin has also been discovered in non-dental tissues, indicating of an even wider function of the protein. An extract from porcine enamel matrix, mainly composed of amelogenin, has been found to have regenerative properties on dental tissues and to improve wound healing, which makes amelogenin interesting from a therapeutic point of view. Amelogenin can also be used to modulate hydroxyapatite formation in vitro, in order to create new improved materials. An increased demand of amelogenin is probable, and especially recombinant amelogenin is of interest since it does not suffer from some of the drawbacks of amelogenin from animal sources, such as risk of viral contamination. This doctoral dissertation presents results from three studies. The main objective with the work has been to improve production of recombinant amelogenin. In the first study it was shown that the expression levels of recombinant murine amelogenin in Escherichia coli are enhanced when an N-terminal histidine tag is fused to the protein. This also leads to an improvement of the growth properties of the host cells, which grow to higher cell densities. The histidine tagged amelogenin can be purified using immobilized metal ion affinity chromatography (IMAC). In the second study a new method for purification of recombinant human amelogenin is described. By heat treating amelogenin expressing E. coli cells at low pH the cells can be disrupted, which releases soluble amelogenin into the surrounding solution. The host cell proteins denatures by the same treatment and form a precipitate. By separating the soluble fraction from the precipitate and cell debris a more than 95 % pure amelogenin solution is obtained. The method combines cell lysis with separation of amelogenin from the host cell proteins, and significantly improves the yield compared to previously used methods. In the third study the proteolytic processing of human amelogenin by matrix metalloproteinase-20 (MMP-20) was examined. Cleavage of amelogenin generates a mixture of different amelogenin polypeptides, similar to what is found in the enamel matrix. The identified MMP-20 cleavage sites on human amelogenin were located in the C- and N-terminus, similar to what has been previously reported for porcine and murine amelogenin. We found that MMP-20 can be permanently inactivated by heat treatment at low pH. This makes it possible to generate amelogenin preparations, proteolytically degraded to different degrees, by terminating the cleavage reaction at a certain point. The results presented in this doctoral dissertation provide improvements to the field that are useful for production of recombinant amelogenin and for future study of amelogenin function.