Analogous effects of recombinant human full-length amelogenin expressed by Pichia pastoris yeast and enamel matrix derivative in vitro
Correspondence: R. Shu, Department of Periodontology, Shanghai Key Laboratory of Stomatology, 9th People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China. Tel.: +86 21 23271241; Fax: +86 21 63135412; E-mail: firstname.lastname@example.org
Amelogenins are proposed to be responsible for enamel matrix derivative (EMD)-induced periodontal regeneration; however, heterogeneity of amelogenins makes it challenging to purify the full-length proteins. This study has been carried out to express and purify a recombinant full-length human amelogenin protein (rHhAm175) in the eukaryotic yeast Pichia pastoris, and further compare biological responses of human periodontal ligament fibroblasts (PDLFs) to rHhAm175 and porcine EMD (pEMD).
Materials and methods
Human cDNA encoding a 175-amino acid amelogenin was subcloned into the pPIC3.5K vector. The rHhAm175 expressed in P. pastoris GS115 (Mut+) was purified and characterized. We examined cell attachment, migration and proliferation responses of human PDLFs to rHhAm175 and pEMD respectively, and characterized associated changes of proliferation-related intracellular signalling molecules, including extracellular signal response kinase (ERK) and Akt kinases/protein kinase B (Akt/PKB) kinases.
The purified rHhAm175 was confirmed to be molecular mass 22 021.13 Da, phosphorylated human amelogenin, and alone significantly promoted proliferation and migration of human PDLFs to an extent comparable to that of pEMD. Cell attachment was increased over the first 60 min incubation with rHhAm175 or pEMD. Both rHhAm175 and pEMD induced PDLF mitogenesis via extracellular signal response kinase (ERK1/2), but not by Akt kinases/protein kinase B (Akt/PKB).
rHhAm175 modulated cell activities of human PDLFs, to a comparable extent as porcine EMD. These data suggest that rHhAm175 might be used to induce periodontal tissue regeneration.