One-Step Purification of Recombinant Human Amelogenin and Use of Amelogenin as a Fusion Partner

Amelogenin is an extracellular protein first identified as a matrix component important for formation of dental enamel during tooth development. Lately, amelogenin has also been found to have positive effects on clinical important areas, such as treatment of periodontal defects, wound healing, and bone regeneration. Here we present a simple method for purification of recombinant human amelogenin expressed in Escherichia coli, based on the solubility properties of amelogenin. The method combines cell lysis with recovery/purification of the protein and generates a >95% pure amelogenin in one step using intact harvested cells as starting material. By using amelogenin as a fusion partner we could further demonstrate that the same method also be can explored to purify other target proteins/peptides in an effective manner. For instance, a fusion between the clinically used protein PTH (parathyroid hormone) and amelogenin was successfully expressed and purified, and the amelogenin part could be removed from PTH by using a site-specific protease

Recombinant amelogenin - Strategies for purification and protein processing

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

In vitro preparation of amelogenin nanoparticles carrying nucleic acids.

Amelogenin, a matrix protein involved in biomineralization of enamel, can self-assemble to form nanospheres in a pH-dependent manner. Nucleic acids (single-stranded, double-stranded, and plasmid DNA, as well as RNA) could be co-precipitated with amelogenin, demonstrating a strong binding of nucleic acids to amelogenin. The amounts of co-precipitated nucleic acids were analyzed and binding levels upto 90 μg DNA/mg amelogenin was achieved. The co-precipitation could also be carried out in a bacterial cell homogenate, and no bacterial proteins were found in the amelogenin aggregates, suggesting specificity for nucleic acid binding. Dynamic light scattering showed that amelogenin nanosphere structure is maintained upon DNA binding with an upto 2.6 nm increase in diameter. The reported binding of nucleic acids to amelogenin can be explored practically for nucleic acid separation

SDS-PAGE analysis of samples from different stages of purification of recombinant amelogenin from <i>E. coli</i> cells.

<p>Lanes marked with (A) represent samples from cells expressing amelogenin and lanes marked with (N) refers to negative control cells harboring empty expression vector. Lanes marked with (1) represent whole cell samples after cultivation. Lanes marked (2), (3) and (4) represent samples after different treatments of the intact cells. (2): soluble fractions of cells sonication in 3% HAc. (3): soluble fractions after heat treatment of the sonicated samples in lanes 2A and 2N. (4): soluble fractions of cells directly heat treated at 80°C. This generates the most pure amelogenin. Lanes marked (M) contain molecular weight markers.</p

Characterization and assembly of a GFP-tagged cylindriform silk into hexameric complexes.

Spider silk has been studied extensively for its attractive mechanical properties and potential applications in medicine and industry. The production of spider silk, however, has been lagging behind for lack of suitable systems. Our approach focuses on solving the production of spider silk by designing, expressing, purifying and characterizing the silk from cylindriform glands. We show that the cylindriform silk protein, in contrast to the commonly used dragline silk protein, is fully folded and stable in solution. With the help of GFP as a fusion tag we enhanced the expression of the silk protein in Escherichia coli and could optimize the downstream processing. Secondary structures analysis by circular dichroism and FTIR shows that the GFP-Silk fusion protein is predominantly α-helical, and that pH can trigger a α- to β-transition resulting in aggregation. Structural analysis by small angle x-ray scattering suggests that the GFP-Silk exists in the form of a hexamer in solution

Histidine tag fusion increases expression levels of active recombinant amelogenin in Escherichia coli

Amelogenin is a dental enamel matrix protein involved in formation of dental enamel. In this study, we have expressed two different recombinant murine amelogenins in Escherichia coli: the untagged rM179, and the histidine tagged rp(H)M180, identical to rM179 except that it carries the additional N-terminal sequence MRGSHHHHHHGS. The effects of the histidine tag on expression levels, and on growth properties of the amelogenin expressing cells were studied. Purification of a crude protein extract containing rp(H)M 180 was also carried out using IMAC and reverse-phase HPLC. The results of this study showed clearly that both growth properties and amelogenin expression levels were improved for E coli cells expressing the histidine tagged amelogenin rp(H)M 180, compared to cells expressing the untagged amelogenin rM179. The positive effect of the histidine tag on amelogenin expression is proposed to be due to the hydrophilic nature of the histidine tag, generating a more hydrophilic amelogenin, which is more compatible with the host cell. Human osteoblasts treated with the purified rp(H)M 180 showed increased levels of secreted osteocalcin, compared to untreated cells. This response was similar to cells treated with enamel matrix derivate, mainly composed by amelogenin, suggesting that the recombinant protein is biologically active. Thus, the histidine tag favors expression and purification of biologically active recombinant amelogenin. (c) 2006 Elsevier Inc. All rights reserved

Amyloid-like ribbons of amelogenins in enamel mineralization

Enamel, the outermost layer of teeth, is an acellular mineralized tissue that cannot regenerate; the mature tissue is composed of high aspect ratio apatite nanocrystals organized into rods and inter-rod regions. Amelogenin constitutes 90% of the protein matrix in developing enamel and plays a central role in guiding the hierarchical organization of apatite crystals observed in mature enamel. To date, a convincing link between amelogenin supramolecular structures and mature enamel has yet to be described, in part because the protein matrix is degraded during tissue maturation. Here we show compelling evidence that amelogenin self-assembles into an amyloid-like structure in vitro and in vivo. We show that enamel matrices stain positive for amyloids and we identify a specific region within amelogenin that self-assembles into β-sheets. We propose that amelogenin nanoribbons template the growth of apatite mineral in human enamel. This is a paradigm shift from the current model of enamel development