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Expression and Activities Experiment of DNA Transduction Motif Based on GAL4 in Pichia Pastoris


  The genes encoding DNA-binding domain(BD) designed based on the yeast transcriptional activator GAL4 and protein transduction domain of HIV-1 Tat protein were fused via soft linker peptide sequence,and cloned into yeast expression vector pPIC9k.The resulted plasmid pTG was linearized and transfected into Pichia pastoris strains GS115 by electroporation.High copies of transformants were obtained with Muts and HIS+ phenotype identification,PCR amplification and screening of G418.After flask culture and expression induced by methanol,the target protein named TG was well expressed and analyzed by SDS-PAGE and Western blot.Under optimized conditions,the yield of soluble recombinant protein was approximately 39.7 mg/L.DNA binding activity and cell transduction property of TG were analyzed by gel electrophoresis and fluorescent microscopy.The results show that the recombinant protein could bind strongly to the plasmid containing upstream activating sequence(UAS).The cell experiments revealed that TG could deliver the binding plasmid into HEK-293 cells effectively.In summary,the work presented here suggests that TG is specific toward UAS containing plasmid and has the potential for use as nonviral DNA delivery agent.……