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Choosing the right baculovirus gene promoter for recombinant protein production

Updated: Nov 20, 2023

The polyhedrin gene promoter is the general work horse used for protein expression in the baculovirus system. It is active in the very late phase of virus replication, after budded virus production, so the peak of recombinant protein production is less likely to affect formation of infectious virions. The p10 gene promoter is also a very late promoter and can give good yields but is slightly weaker than polyhedrin. It is often used in combination with polyhedrin in dual expression vectors (e.g. pOET5.1).

In contrast, the p6.9 gene promoter is an element active in the late phase of gene expression (i.e. just before polyhedrin and p10). It is not as strong as those two promoters but can sometimes give better yields of protein, particularly with secreted proteins (see references below). Unlike polyhedrin and p10, the p6.9 encodes a protein essential for virus replication. It has a highly basic nature, which is required for its role in condensation of virus DNA prior to incorporation into capsid structures. The capsids then bud from infected cells, acquiring a lipoprotein envelope, which also incorporates GP64, a protein essential for virus uptake into susceptible cells. P6.9 is sometimes referred to as the basic protein.

The p6.9 gene cannot be deleted from the baculovirus genome without affecting virus formation. Therefore, to use it as an expression vector requires that it is copied into another location in the virus genome. Typically, this is done by replacing the polyhedrin gene promoter in say pOET1.1 with the p6.9 gene promoter to yield either pOET3 or pOET4.

The reasons for the superiority of the p6.9 promoter for producing some recombinant proteins are unclear. The ones that are expressed to a higher level than with polyhedrin promoters tend to be those that are secreted from cells. It may be that in the late phase of expression that the secretory pathway of the cell is in better shape than in the subsequent very late phase.

Alternatively, the great burst of protein production in the very late phase may simply overwhelm the endoplasmic reticulum and cause a logjam in secretion. In the late phase of expression, using p6.9, it could be a case of little less results in better overall yield.

Bonning et al. (1994). Superior expression of juvenile hormone esterase and β-galactosidase from the basic protein promoter of Autographa californica nuclear polyhedrosis virus compared to the p10 protein and polyhedrin promoters.

Lawrie et al. (1995). High level synthesis and secretion of human urokinase using a late gene promoter of the Autographa californica nuclear polyhedrosis virus.

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