The Expression,Separation And Purification Of RhCEMP1with The Eukaryotic Expression Vector PWX530in Yeast

Recently a novel human cementum protein isoform has been cloned from a human cementoblastoma-derived cell line, which has been named cemetum protein1(CEMP1). Research data shows that the expression of CEMP1is limited to cementoblasts,may be play an important role in periodontal ligament cells differentiation,in other words,CEMPl is a biological marker of cemetum.In order to understand the function of CEMP1,researchers find that CEMP1can promote cell adhesion and differentiation, plays a role during the biomineralization process by promoting octacalcium phosphate (OCP) crystal nucleation,also the expression of CEMP1induces mineralization phenotype and promotes calcium deposits in nonosteogenic cells.all these results imply that CEMP1might have a potential function in cementum formation and could be used for achieveing physiological dental implant.This study planed to construct the recombinant eukaryotic expression vector containing the human cementum protein1by bio-engineering technology/the vector was transfectd into yeast for expression,then got CEMP1from yeast condition medium.Part1Construction of a eukaryotic expression vector of recombination human cementum protein1Objective:To construct the recombinant eukaryotic expression vector containing the recombination human cementum protein1(rhCEMP1).Methods:rhCEMPl cDNA was amplified by PCR, and correctly inserted into corresponding sites of intermediate vector pTeasy after restriction endonuclease digestion, and then inserted into corresponding sites of eukaryotic expression vertor pWX530after another restriction endonuclease digestion. The recombinant vector pWX530-rhCEMPl was confirmed to contain rhCEMP1DNA sequence by agarose gel electrophoresis and DNA sequence analysis.Results:The construction of the recombinant eukaryotic expression vector pWX530-rhCEMPl and the correct DNA sequence were confirmed through restriction enzyme maping analysis and DNA sequencing.Conclusions:The recombinant vector pWX530-rhCEMPl was constructed successfully.Part2The expression, separation and purification of rhCEMPl in yeast Methods:The vector pWX530-rhCEMPl was transfectd into yeast competent cells, the yeast was cultured after amino acid auxotroph screening. The expression of rhCEMP1was determined by SDS-PAGE and enzyme-linked immunosorbent assay(ELISA),the rhCEMP1was purified by ion exchange chromatography.Results:The recombinant plasmid was successfully transferred to the yeast cells. The rhCEMP1was successful expressed by SDS-PAGE and ELISA analysis.Conclusions:the recombinant plasmid pWX530-rhCEMP1could be transfected into yeast and expressed.