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

2.4. Modification of a dual expression vector, pFastBac Dual 
To improve this dual vector, the AcMNPV 
polh
pA sequence was first cloned into 
pFastBac Dual. This was achieved by a digestion of pFastBac-M2 and pFastBac Dual separately 
with HindIII and EcoRV. The DNA fragments were separated by agarose gel electrophoresis.

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The 3,374 bp HindIII/EcoRV fragment that contained the 
polh
pA sequence and the 1,774 bp 
HindIII/EcoRV fragment that contained the MCS and the 
p10
promoter were gel purified and 
ligated for the production of pFastBac-Dual-M1 (Fig. 1C).
To insert the 80 bp 
cis 
element into pFastBac-Dual-M1, the plasmid pFastBac-M2 was 
digested with SnaBI and BamHI, and pFastBac-Dual-M1 was digested with BstZ17I and BamHI, 
followed by agarose gel electrophoresis. The 201 bp fragment containing the 80 bp 
cis
element 
and the 50 bp 
polh 
promoter from the pFastBac-M2 digestion, plus the larger fragment from the 
pFastBac-Dual-M1 digestion, were gel purified and ligated for the production of pFastBac-Dual-
M2 (Fig. 1C). To further confirm the significance of the 80 bp 
cis
element in enhancing the 50 
bp 
polh
promoter activity, the 224 bp 
polh
upstream fragment in pFastBac-M1 was retrieved by a 
digestion of SnaBI and BamHI and ligated into the BstZ17I and BamHI sites of pFastBac-Dual-
M1 by T4 DNA ligase (NEB) to produce pFastBac-Dual-M3 (Fig. 1C). 
2.5. Cytoplasmic particle purification and identification 
High Five cells at 5×10
5
cells per 35 mm tissue culture dish were infected with AcP3, 
AcBac-PolhE, AcBac-PolhED or AcSDP33-35 at an MOI of 1. At day 3 post-infection (P.I.) or 
when cytoplasmic particles appeared, the medium was removed and 1% SDS was added to lyse 
the cells. The lysates were filtered with a Whatman Nuclepore Track-Etch membrane (pore size 
8 µm). Particles retained on the membrane were washed with TE in 1.5 ml microcentrifuge 
tubes. An aliquot from each infection was examined under a microscope for the purity of 
cytoplasmic particles. To determine the nature of these cytoplasmic particles, the purified