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 between insulin-like growth factors (IGFs) and cytokines is described. The cDNA of BOMIGF, a fusion protein between the insect insulin-like peptide bombyxin and IGF II, has been linked to the gene of interleukin-3. The BOMIGF-interleukin 3 fusion gene was cloned downstream of the promoter regions of the p10 and polyhedrin proteins within baculovirus transfer 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 Trichoplusia ni (BTI-TN-5B1-4) were infected with the recombinant baculoviruses obtained from the three transfer vectors. Trichoplusia ni cells produced the largest amount of recombinant protein. Although the efficiency of the three recombinant viruses was remarkably similar, the baculovirus with the gene present behind both promoters produced relatively more recombinant protein in host cells than those viruses driven with the polyhedrin or p10 promoters alone. The BOMIGF-interleukin-3 chimera was stable and continuously increased in the culture medium up to 5-6 days postinfection. Therefore the addition of a protease inhibitor was useful only at the stage of massive host cell death. Medium supplemented with copper sulfate was detrimental for the long-term production of the fusion protein.
DiFalco
M R
MR
Endocrine Laboratory, Royal Victoria Hospital, Montreal, Canada.
Bakopanos
E
E
Patricelli
M
M
Chan
G
G
Congote
L F
LF
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D007377:Interleukin-3 / Q000096:biosynthesis*; D000077183:Occlusion Body Matrix Proteins; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D013002:Somatomedins / Q000096:biosynthesis*; D018411:Spodoptera; D014764:Viral Proteins / Q000235:genetics*; D015678:Viral Structural Proteins
D007377:Interleukin-3; D000077183:Occlusion Body Matrix Proteins; D011993:Recombinant Fusion Proteins; D013002:Somatomedins; D014764:Viral Proteins; D015678:Viral Structural Proteins; C009829:polyhedrin protein, Nucleopolyhedrovirus