【摘要】 根据天然拖丝蛋白的高度重复性序列,引入与细胞黏附有关的精氨酸-甘氨酸-天冬氨酸(RGD)三肽序列,以毕赤酵母偏好的密码子化学合成RGD-拖丝蛋白基因单体,通过"头尾相连"的多聚化策略,倍加成16聚体与32聚体基因.分别将这两种多聚体与分泌型表达载体pPIC9K连接,转化毕赤酵母GS115,用G418筛选毕赤酵母重组菌.通过甲醇诱导表达,培养液上清的SDS-PAGE分析表明RGD-重组拖丝蛋白获得分泌型表达.更多还原

【Abstract】 Based on the high repetitive sequence of natural spider dragline silk and with the introduced RGD peptide condons involved in cell adhesion,the DNA monomer sequence was synthesized by using preferred condons of Pichia pastoris.The monomer was multimized to get 16-mer and 32-mer by the construction strategy " the head to tail ".The two multimers were ligated into secretory expression vector pPIC9K.The constructs were transformed into Pichia pastoris strain GS115.The multicopy integrants were screened on medium containing increasing concentrations of G418 and induced by methanol.SDS-PAGE analysis of superatants of expression products demonstrated that RGD-spider dragline silk could be expressed secretorily in Pichia pastoris.更多还原