THE INFLUENCE OF VARIOUS INSECT-CELL LINES, P10 AND POLYHEDRIN PROMOTERS IN THE PRODUCTION OF SECRETED INSULIN-LIKE GROWTH FACTOR-INTERLEUKIN-3 CHIMERAS IN THE BACULOVIRUS EXPRESSION SYSTEM

A technique for the optimal synthesis of secreted fusion proteins betw een insulin-like growth factors (IGFs) and cytokines is described. The cDNA of BOMIGF, a fusion protein between the insect insulin-like pept ide bombyxin and IGF II, has been linked to the gene of interleukin-3. The BOMIGF-interleukin 3 fusion gene was cloned downstream of the pro moter regions of the p10 and polyhedrin proteins within baculovirus tr ansfer vectors. A third, dual transfer vector was constructed with the gene inserted simultaneously behind p10 and polyhedrin promoters. Two different lepidopteran cell lines, Spodoptera frugiperda (Sf9) and Tr ichoplusia ni (BTI-TN-5B1-4) were infected with the recombinant baculo viruses obtained from the three transfer vectors. Trichoplusia ni cell s produced the largest amount of recombinant protein. Although the eff iciency of the three recombinant viruses was remarkably similar, the b aculovirus with the gene present behind both promoters produced relati vely more recombinant protein in host cells than those viruses driven with the polyhedrin or p10 promoters alone. The BOMIGF-interleukin-3 c himera was stable and continuously increased in the culture medium up to 5-6 days postinfection. Therefore the addition of a protease inhibi tor was useful only at the stage of massive host cell death. Medium su pplemented with copper sulfate was detrimental for the long-term produ ction of the fusion protein. (C) 1997 Elsevier Science B.V.