A new method for protein coexpression in Escherichia coli using two incompatible plasmids

It is commonly believed that incompatible plasmids carrying the same replic on cannot coexist stably in one Escherichia coli cell. However, we found th at two incompatible plasmids carrying different antibiotic resistance genes , if under the selection pressure of the two antibiotics, can coexist in E. coli for at least 14 h, which is adequate for routine culture and protein expression. Based on this discovery, we developed a new method to coexpress foreign proteins in E. coli using two incompatible plasmids. The coding re gions of the two subunits (DFF45 and DFF40) of the human DNA fragmentation factor (DFF) were cloned into two incompatible bacterial expression vectors -pET-21a with ampicillin resistance and pET-28a with kanamycin resistance, respectively. The two resulting plasmids were used to cotransform E. coli B L21(DE3) cells. After selection by ampicillin and kanamycin simultaneously, cotransformants that contain both recombinant plasmids were obtained. Indu ced by isopropyl beta -D-thiogalactoside, DFF45, and DFF40 were coexpressed efficiently in the presence of the two antibiotics. The coexpression produ ct contained adequate soluble portions for both DFF45 and DFF40, while all DFF40 was insoluble if expressed alone. The coexpression product also exhib ited the same caspase-activated DNase activity as its natural counterparts, which cannot be obtained if its two subunits are expressed separately. (C) 2001 Academic Press.