Donor plasmid vectors
AssigneeMiami University
InventorsXiao-Wen Cheng, Hui Shang, Tyler Garretson
Life of this patent
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Abstract
Certain donor plasmid vectors such as pFastBac™1 and pFastBac™ Dual lack a cis DNA element upstream of the polh translation start codon (ATG) present in wild type (wt) Autographa californica multiple nucleopolyhedrovirus (AcMNPV), and contain a SV40 pA fragment. When a cis DNA element is inserted upstream of the 50 bp polh promoter and SV40 pA was replaced with a AcMNPV polh pA signal in pFastBac™1 and pFastBac™Dual, certain protein expression levels in High Five™ cells using the Bac-to-Bac® system reached that of the wt AcMNPV.
Claims4
A method of improving protein expression in a cell capable of being infected by AcMNPV comprising:
- (i) providing a first AcMNPV sequence that has been modified to comprise a heterologous nucleic acid encoding a cis element having 95% to 100% identity to SEQ ID NO:1, a polh promoter, a polh ORF, and a polh pA signal sequence;
- wherein the cis element is located 147 bp upstream of the polh promoter to form a first modified AcMNPV bacmid, and
- (ii) infecting the cell with the first modified AcMNPV bacmid, wherein the cell infected with the first modified AcMNPV bacmid expresses at least 3 times the level of polyhedrin protein as compared to a cell infected with a second AcMNPV bacmid comprising a SV40 pA signal sequence.
A method of improving protein expression in a cell capable of being infected by AcMNPV comprising:
- (i) modifying a first AcMNPV sequence to replace a SV40 pA signal sequence with an AcMNPV polh pA signal sequence to form a first modified AcMNPV bacmid, and
- (ii) infecting the cell with the first modified AcMNPV bacmid, wherein the cell infected with the first modified AcMNPV bacmid expresses increased levels of polyhedrin protein as compared to a cell infected with a second AcMNPV bacmid that still comprises the SV40 pA signal sequence.
Dependencies are inferred from the claim text.