AbstractThe Burst of expression from polyhedrin (polh) promoter during very late phase of baculovirus infection requires a sequence located between TAAG and the translation initiation site, typically referred to as burst sequence (BS). The expression of polh promoter is stimulated by specifically binding of very late transcriptional factor 1 (VLF‐1) to BS. In order to enhance the production of recombinant proteins the polh promoter was modified via a multiple BS bacmid system in which the number of BSs was increased. Compared to an expression from a normal polh promoter, β‐glucuronidase (GUS) activity in High Five insect cells was three times higher with a modified polh promoter containing two BSs. Using a modified polh promoter that contains nine BSs in silkworm expression system, β1‐3‐N‐acetylglucosaminyltransferase 2 (β3GnT2) activity per larva was 6.8‐fold higher than control. Furthermore, the co‐expression of modified promoters along with VLF‐1‐enhanced β3GnT activity. Thus, an increased optimal number of BS and its co‐expression with VLF‐1 leads to the production of higher level of gene expression in insect cells and silkworm larvae. This new modified promoter engineered in the current study is the strongest promoter for overexpressing foreign proteins in an eukaryotic cell and system, thus leading a progress in baculovirus–insect cell and silkworm biotechnology. Biotechnol. Bioeng. 2010;107: 909–916. © 2010 Wiley Periodicals, Inc.