Virus Research (Nov 2022)
Homologous recombination risk in baculovirus expression vector system
Abstract
The baculovirus expression vector system (BEVS) is widely used for producing recombinant proteins. To achieve high expression level of recombinant proteins, baculoviral elements, such as enhancers, promoters, signal peptide coding sequences and 3′-UTR, have been extensively employed. There is a recombination risk derived from homologous sequences between viral genome and functional baculovirus-derived elements associated with foreign genes. Although homologous recombination have distinct biological functions, these potential adverse recombination may trigger a DNA fragment being inverted or looped out, resulting in the production of defective viruses and eventual yields declines of recombinant proteins. However, the risk of such homologous recombination has not been systematically assessed. Here, we measured the recombination rate using a promoter-less fluorescent reporter integrated with various lengths homologous of p10 coding region. Homologous fragments longer than 60 bp possess sufficient recombination probability and exerts effect on purity and integrity of virus. Shortening the length of homologous fragments and separating homologous fragments by point mutations can effectively reduce unfavorable recombination. These findings reveal a homologous recombination risk resulted from genome-homologous baculoviral elements and propose reliable strategies reducing recombination rate to facilitate viral stability and integrity in baculovirus expression vector system.
