High yield of biologically active recombinant human amelogenin using the baculovirus expression system.
The amelogenins are secreted by the ameloblast cells of developing teeth; they constitute about 90% of the enamel matrix proteins and play an important role in enamel biomineralization. Recent evidence suggests that amelogenin may also be involved in the regeneration of the periodontal tissues and that different isoforms may have cell-signalling effects. During enamel development and mineralization, the amelogenins are lost from the tissue due to sequential degradation by specific proteases, making isolation of substantial purified quantities of full-length amelogenin challenging. The aim of the present study was to express and characterize a recombinant human amelogenin protein in the eukaryotic baculovirus system in quantities sufficient for structural and functional studies. Human cDNA coding for a 175 amino acid amelogenin protein was subcloned into the pFastBac HTb vector (Invitrogen), this system adds a hexa-histidine tag and an rTEV protease cleavage site to the amino terminus of the expressed protein, enabling effective one-step purification by Ni2+-NTA affinity chromatography. The recombinant protein was expressed in Spodoptera frugiperda (Sf9) insect cells and the yield of purified his-tagged human amelogenin (rHAM+) was up to 10 mg/L culture. Recombinant human amelogenin (rHAM+) was characterized by SDS-PAGE, Western blot, ESI-TOF spectrometry, peptide mapping, and MS/MS sequencing. Production of significant amounts of pure, full-length amelogenin opened up the possibility to investigate novel functions of amelogenin. Our recent in vivo regeneration studies reveal that the rHAM+ alone could bring about regeneration of the periodontal tissues; cementum, periodontal ligament, and bone.
Taylor
Angela L
AL
Dental Research Laboratory, Institute of Dental Sciences, Hebrew University-Hadassah Faculty of Dental Medicine, Hebrew University, Jerusalem, Israel.
Haze-Filderman
Amir
A
Blumenfeld
Anat
A
Shay
Boaz
B
Dafni
Leah
L
Rosenfeld
Eli
E
Leiser
Yoav
Y
Fermon
Eran
E
Gruenbaum-Cohen
Yael
Y
Deutsch
Dan
D
D053523:Amelogenin; D000818:Animals; D016367:Baculoviridae / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D003746:Dental Enamel Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D020224:Expressed Sequence Tags; D006801:Humans; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D018411:Spodoptera / Q000378:metabolism
D053523:Amelogenin; D003746:Dental Enamel Proteins; D011994:Recombinant Proteins