High-level expression and improved folding of proteins by using the vp39 late promoter enhanced with homologous DNA regions.
Some recombinant proteins expressed by baculovirus expression vector systems (BEVS) aggregate because the BEVS can produce large amounts of protein late during infection, when post-translational modification and protein quality control mechanisms are inactive. For expression during earlier stages than that driven by the polyhedrin (polh) very late promoter, transfer vectors were generated in which this promoter was replaced with a green fluorescent protein (GFP) gene controlled by a vp39 late promoter modified to contain HR3, one of the homologous DNA regions (HRs) of Bombyx mori nuclear polyhedrosis virus (BmNPV). The rise times of the fluorescence of GFP expressed by using recombinant viruses carrying the modified vp39 promoter were earlier than those associated with either the polh promoter or the native vp39 promoter lacking HR3. In transient expression assays, the vp39 late promoter in transfer vectors behaved like a delayed-early promoter, and was enhanced by HR3, and required IE-1 protein and various viral gene products encoded on both sides of BmNPV polh. When the vp39 promoter with HR3 was used, the aggregation of several foreign proteins expressed by the BEVS was markedly decreased. This study provides a new option for the expression of sufficiently quality-controlled proteins by using the vp39 promoter and HR3 in BEVS early in baculovirus infection, when the infection has caused little damage in the host cells.
Ishiyama
Seiji
S
Research Institute of Biological Science, KATAKURA Industries, Co., Ltd., 1548 Shimo-okudomi, Sayama, Saitama 350-1332, Japan. s.ishiyama@katakura.co.jp
Ikeda
Masahiro
M
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D012831:Bombyx; D002460:Cell Line; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D017510:Protein Folding; D011994:Recombinant Proteins / Q000378:metabolism*
D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins