Construction and Expression of Recombinant Pichia pastoris for pPIC3.5K/Thanatin-( G8S2)-PPA

The fusion gene of thanatin-linker-ppa is cloned into the Pichia pastoris expression vector.The pPlC3.5K/Thanatin-(G8S2)-PPA yeast expression vector is constructed and then sequenced.The pPIC3.5K/Thanatin-(G8S2)-PPA is linearized by Sal I enzyme,and transformed into GS115 Pichia pastoris using electricity.Then the transformants are identified by PCR and selected with high G418 resistance on a YPD plate.The positive Pichia pastorist transformants of pPlC3.5K/Thanatin-(G8S2)-PPA are induced by methynol.SDS-PAGE analysis and western bloting show that fusion protein is attained,and its molecular weight is about 31 kD.The anti-tumor activity of fusion protein,which is purified by Ni-NTA affinity chromatography,is analyzed by MTT,and the results indicate that fusion protein exhibits significant antitumor activity.