Improved pFastBac™ donor plasmid vectors for higher protein production using the Bac-to-Bac® baculovirus expression vector system

•Bac-to-Bac expresses lower protein levels than wild type AcMNPV.•The transfer vectors of Bac-to-Bac lack an 85 bp cis element and contain SV40 pA.•The polyhedrin pA is required for higher protein expression levels.

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-based Bac-to-Bac® expression system consists of a bacmid and five pFastBac™ donor transfer vectors. It has been widely used for eukaryotic gene expression in insect cells to elucidate gene function in biotechnology laboratories. The pFastBac™ vectors contain a 50 bp AcMNPV polyhedrin (polh) promoter and a 127 bp SV40 polyadenylation (pA) signal for cloning a gene of interest into the bacmid, resulting in unsolved lower gene expression levels than the wild type (wt) AcMNPV in insect cells. Therefore, the purpose of this research is to understand why the Bac-to-Bac system produces lower gene expression levels. Here, we determined that bacmids transposed with pFastBac™ vectors produced 3-4 fold lower levels of certain proteins than the wt AcMNPV. We found that an 80 bp cis element 147 bp upstream of the 50 bp polh promoter and a 134 bp polh pA signal are required in pFastBac™ to achieve bacmid protein expression levels equivalent to wt AcMNPV in High Five insect cells. Therefore, researchers currently using pFastBac™ vectors for protein expression can transfer their genes of interest into the improved vectors in this report to elevate protein expression yields in insect cells to reduce protein production costs.