Analogous effects of recombinant human full-length amelogenin expressed by Pichia pastoris yeast and enamel matrix derivative in vitro
Analogous effects of recombinant human full-length amelogenin expressed by Pichia pastoris yeast and enamel matrix derivative in vitro
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DOI：
10.1111/j.1365-2184.2012.00834.x
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发表时间：
2012-10-01
影响因子：
8.5
通讯作者：
Tian, L.
中科院分区：
文献类型：
--
作者：
Cheng, L.;Lin, Z. K.;Tian, L.
目的釉原蛋白是釉基质衍生物（elaminmatrix derivative，EMD）诱导的牙周组织再生的重要蛋白质，但其异质性使得全长蛋白质的纯化具有挑战性。本研究旨在利用毕赤酵母表达和纯化人釉原蛋白（rHhAm 175），并比较人牙周膜成纤维细胞（PDLFs）对rHhAm 175和猪EMD（pEMD）的生物学反应。材料和方法将编码175个氨基酸的釉原蛋白的人cDNA亚克隆到pPIC 3.5K载体中。对在毕赤酵母GS115（Mut+）中表达的rHhAm 175进行了纯化和表征。我们分别检测了人PDLFs对rHhAm 175和pEMD的细胞粘附、迁移和增殖反应，并表征了增殖相关的细胞内信号分子的相关变化，包括细胞外信号应答激酶（ERK）和Akt激酶/蛋白激酶B（Akt/PKB）激酶。结果纯化的rHhAm 175分子量为22 021.13 Da，为磷酸化的人釉原蛋白，单独使用可显著促进人牙周膜成纤维细胞的增殖和迁移，其作用与pEMD相当。在与rHhAm 175或pEMD孵育的前60分钟内，细胞附着增加。rHhAm 175和pEMD均通过细胞外信号应答激酶（ERK 1/2）诱导PDLF有丝分裂，而不通过Akt激酶/蛋白激酶B（Akt/PKB）诱导。结论rHhAm 175对人PDLFs细胞活性的调节作用与猪EMD相当。这些数据表明，rHhAm 175可能用于诱导牙周组织再生。
Objectives 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. Results 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). Conclusions 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.