Expression of soybean Kunitz trypsin inhibitor gene SKTI in Dunaliella salina

Recently, microalgae have attracted much attention as a new bioreactor system for producing high-value heterologous proteins. In this paper, we investigated the expression of soybean Kunitz trypsin inhibitor gene SKTI in the chlorophyte Dunaliella salina. Using D. salina genomic DNA as template, the entire coding region of SKTI was amplified by PCR as a 654-bp DNA fragment. It had 100 % identity with the published sequence of SKTI. The entire SKTI fragment was cloned into the expression vector pCAM2201 at a site just downstream of 35S promoter of to yield pCAMSKTI. D. salina cells transfected with pCAMSKTI by means of the lithium acetate/polyethylene glycol-mediated method expressed a protein of 20.1 kDa as detected by anti-SKTI antibody. In addition, SDS-PAGE analysis of the cell extract also revealed an intense protein band indicative of the recombinant SKTI. SKTI was therefore successfully expressed in D. salina, and the expression could be detected for at least 35 generations.