Expression of human soluble TRAIL in Chlamydomonas reinhardtii chloroplast

Tumor necrosis factor-related apop-tosis-inducing ligand (TRAIL) induces selectively apoptosis in various tumor cells and virus-infected cells, but rarely in normal cells. A chloroplast expres-sion vector, p64TRAIL, containing the cDNA coding for the soluble TRAIL (sTRAIL), was constructed with clpP-trnL-petB-chlL-rpl23-rpl2 as Chlamydomonas reinhardtii plastid homologous recombinant frag-ments and spectinomycin-resistant aadA gene as a select marker. The plasmid p64TRAIL was trans-ferred into the chloroplast genome of C. reinhardtii by the biolistic method. Three independently trans-formed lines were obtained by 100 mg/L spectino-mycin selection. PCR amplification, Southern blot analysis of the sTRAIL coding region DNA and culti-vation cells in the dark all showed that the exogenous DNA had been integrated into chloroplast genome of C. reinhardtii. Western blot analysis showed that human soluble TRAIL was expressed in C. reinhardtii chloroplast. The densitometric analysis of Western blot indicated that the expressed human sTRAIL protein in the chloroplasts of C. reinhardtii accounted for about 0.43%―0.67% of the total soluble proteins. These experimental results demonstrated the possi-bility of using transgenic chloroplasts of green alga as bioreactors for production of biopharmaceuticals.