| 0807 Production and Bioactivity of Recombinant Enamelin | ||
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T. IWATA1, J. HU1, Y. YAMAKOSHI1, I. ISHIKAWA2, and J.P. SIMMER1, 1University of Michigan, Ann Arbor, USA, 2Tokyo Medical & Dental University, Japan Enamelin is an extracellular matrix glycoprotein with important functions in enamel formation. Enamelin is rapidly cleaved following its secretion, so the intact protein cannot be isolated from developing enamel, and its structure and biological activities are not well understood. Objectives: to produce and test the bioactivities of enamelin on ameloblast-enriched cultured EOE cells. Methods: Unerupted third molar tooth buds were surgically extracted from six-month-old pigs. The enamel organs were removed and the epithelial cells closest to the enamel layer were obtained by dissecting with a scalpel. These EOE tissues were washed 3x in PBS/gentamicin/fungizone, and dissociated into a suspension of single cells with trypsin/glucose. Passing the suspension through a cell sieve eliminated undissociated tissue. For recombinant expression, amplified porcine enamelin cDNA was ligated into pEF6/V5-His-TOPO, which fused histo-V5-epitope tags onto the enamelin C-terminus. 293-F cells were transfected with the expression construct, and rENAM was isolated using a TALON affinity column and RP-HPLC. Cell adhesion assays were performed in microtiter plates. Wells were coated overnight with increasing concentrations of rENAM, rP172, rAMBN, or type I collagen, blocked with BSA, and incubated with 20,000 ameloblast-enriched EOE cells. The plates were washed with PBS 3x to remove unattached cells. Cell attachment was assayed and quantified using phase contrast microscopy, crystal violet staining, and MTS assay. Results: Recombinant enamelin has an apparent Mr of 160 kDa and tests positive for N-glycosylation. Recombinant enamelin showed cell adhesion activity with ameloblast-enriched EOE cells in a dose dependent manner. The enamelin cell attachment activity was half that of type I collagen, while rP172 and rAMBN showed no cell adhesion activity. Conclusions: N-glycosylated recombinant enamelin was expressed and purified from 293F cells and is positive for EOE-derived cell adhesion activity. This study is supported by NIDCR grant DE11301. | ||
| Seq #70 - Mechanisms of Odontogenesis 11:00 AM-12:00 PM, Thursday, 29 June 2006 Brisbane Convention & Exhibition Centre Exhibit Hall 1 | ||
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