High yield expression of biologically active recombinant full length human tuftelin protein in baculovirus-infected insect cells.
Tuftelin is an acidic protein expressed at very early stages of mouse odontogenesis. It was suggested to play a role during epithelial-mesenchymal interactions, and later, when enamel formation commences, to be involved in enamel mineralization. Tuftelin was also detected in several normal soft tissues of different origins and some of their corresponding cancerous tissues. Tuftelin is expressed in low quantities, and undergoes degradation in the enamel extracellular matrix. To investigate the structure and function of tuftelin, the full length recombinant human tuftelin protein was produced. The full length human tuftelin cDNA was cloned using Gateway recombination into the Bac-to-Bac system compatible transfer vector pDest10. This vector adds a hexahistidine tag to the N-terminus of the expressed protein, enabling one-step affinity purification on nickel column. The recombinant human tuftelin protein was transposed into the bacmid and expressed in Spodoptera frugiperda (Sf9) insect cells. The yield of the purified, his-tagged recombinant full length human Tuftelin (rHTuft+) was 5-8 mg/L culture. rHTuft+ was characterized by SDS-PAGE, Western blot, ESI-TOF spectrometry, restriction mapping and MS/MS sequencing. The availability of the purified, full length recombinant human tuftelin protein opened up the possibility to investigate novel functions of tuftelin. Application of rHTuft+ agarose beads onto embryonic mouse mandibular explants caused changes in the surrounding epithelial cells, including morphology, orientation and spatial organization. Further studies using DiI labeling, revealed that rHTuft+, placed on the tooth germ region, brought about recruitment of adjacent embryonic mesenchymal cells. These findings support the hypothesis that tuftelin plays an important role during embryogenesis.
Shay
B
B
Dental Research Laboratory, Institute of Dental Sciences, Hebrew University-Hadassah Faculty of Dental Medicine, Israel.
Gruenbaum-Cohen
Y
Y
Tucker
A S
AS
Taylor
A L
AL
Rosenfeld
E
E
Haze
A
A
Dafni
L
L
Leiser
Y
Y
Fermon
E
E
Danieli
T
T
Blumenfeld
A
A
Deutsch
D
D
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D002478:Cells, Cultured; D003746:Dental Enamel Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D005260:Female; D006651:Histocytochemistry; D006801:Humans; D008297:Male; D008334:Mandible / Q000187:drug effects / Q000196:embryology / Q000254:growth & development; D013058:Mass Spectrometry; D051379:Mice; D008863:Microspheres; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D018411:Spodoptera / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
D003746:Dental Enamel Proteins; D011994:Recombinant Proteins; C038456:tuftelin