title: Utility of temporally distinct baculovirus promoters for constitutive and baculovirus-inducible transgene expression in transformed insect cells.
abstract: Genetically transformed lepidopteran insect cell lines have biotechnological applications as constitutive recombinant protein production platforms and improved hosts for baculovirus-mediated recombinant protein production. Insect cell transformation is often accomplished with a DNA construct(s) encoding a foreign protein(s) under the transcriptional control of a baculovirus immediate early promoter, such as the ie1 promoter. However, the potential utility of increasingly stronger promoters from later baculovirus gene classes, such as delayed early (39K), late (p6.9), and very late (polh), has not been systematically assessed. Hence, we produced DNA constructs encoding secreted alkaline phosphatase (SEAP) under the transcriptional control of each of the four temporally distinct classes of baculovirus promoters, used them to transform insect cells, and compared the levels of SEAP RNA and protein production obtained before and after baculovirus infection. The ie1 construct was the only one that supported SEAP protein production by transformed insect cells prior to baculovirus infection, confirming that only immediate early promoters can be used to isolate transformed insect cells for constitutive recombinant protein production. However, baculovirus infection activated transgene expression by all four classes of baculovirus promoters. After infection, cells transformed with the very late (polh) and late (p6.9) promoter constructs produced the highest levels of SEAP RNA, but only low levels of SEAP protein. Conversely, cells transformed with the immediate early (ie1) and delayed early (39K) promoter constructs produced lower levels of RNA, but equal or higher levels of SEAP protein. Unexpectedly, the 39K promoter construct provided tightly regulated, baculovirus-inducible protein production at higher levels than the later promoter constructs. Thus, this study demonstrated the utility of the 39K promoter for insect cell engineering, particularly when one requires higher levels of effector protein production than obtained with ie1 and/or when constitutive transgene expression adversely impacts host cell fitness and/or genetic stability.
authors: Department of Molecular Biology, University of Wyoming, 1000 E. University Avenue, Laramie, WY 82071, USA.
authors: Chi-Hung
authors: CH
authors: Lin
authors: Donald L
authors: DL
authors: Jarvis
mesh_terms: D000469:Alkaline Phosphatase / Q000096:biosynthesis* / Q000235:genetics; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D015967:Gene Expression Regulation, Viral; D007313:Insecta / Q000166:cytology / Q000235:genetics* / Q000821:virology; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D019076:Transgenes
chemical_list: D011994:Recombinant Proteins; D000469:Alkaline Phosphatase