关键词: fascin蛋白, 毕赤酵母, 分泌表达, pPIC9载体

Abstract: BACKGROUND AND AIM: To express the human fascin protein, an important tumor molecular marker, in Pichia pastoris. MATERIALS AND METHODS: The recombinant plasmid pPIC9-fascin was constructed by inserting the full coding DNA sequence of fascin into the secreted expression yeast vector pPIC9. Then pPIC9-fascin was linearized by Mss I and transformed into Pichia pastoris GS115 strain. After identification of transformants by PCR, the positive transformant was cultured and induced by 0.5％ methanol. Silver staining and Western blotting were used to analyz the expression level of fascin in the supernatant. RESULTS: Human fascin protein could be expressed in yeast. Silver staining and western blotting analysis from the supernatant showed the molecular weight of fascin protein expressed by the recombinant GS115 strain was about 69 kD, however, the concentration was less than 40 μg/L . CONCLUSION: Although human fascin could be expressed by Pichia pastoris expression system, the low level was far off from that required for bioengineering.

Key words: fascin protein, Pichia pastoris, secreted expression, pPIC9 vector