glyA基因的克隆及其在毕赤酵母中的表达

Cloning of glyA Gene and Its Expression in Pichia pastoris

根据已知的序列设计引物,以大肠杆菌XL10-Gold总DNA为模板进行梯度PCR,并进行DNA序列测定,其序列与已经报道的glyA基因完全一致。将其克隆到毕赤酵母分泌型表达载体pHBM905C上,获得表达质粒pHBM1001.该质粒转化毕赤酵母GS115所得重组子经PCR验证后成功进行了诱导表达,并初步测定了酶活力。

Based on the published sequence, a DNA fragment was cloned by touchdown PCR from E. coli XL10 -Gold. Sequencing showed that this fragment was the same as the published glyA gene. This fragment was inserted into the Pichia pastoris excretion expression vector pHBM905C, and pHBMI001 was obtained. Then the pHBM1001 was introduced into Pichia pastoris GS115, and one clone was selected for induced expression after PCR confirmation, SHMT activity also was obtained.