Flaviviruses, including dengue virus (DENV), West Nile virus, Japanese encephalitis virus, yellow fever virus (YFV) and Zika virus are mosquito-borne viruses responsible for human disease, with significantly different pathogenic outcomes. The flavivirus non-structural protein-1 (NS1) exists in multiple oligomeric forms and has a variety of functions, including involvement in viral replication and virion production within the infected cell. NS1 has also been associated with immune and endothelial cell modulation via a toll-like receptor-4 (TLR4) mediated pathway. To facilitate NS1 functional studies, high expression levels of recombinant protein are required. This is particularly important for TLR4-mediated functional assays, where trace endotoxin contamination can confound experimental outcomes. It was hypothesised that codon optimisation of the NS1 gene would enhance protein yield in a Chinese hamster ovary (CHO) cell protein expression system.
The genetic sequence of DENV and YFV NS1 was modified in silico to synonymously replace rarely utilised codons with those preferentially implemented by CHO cells. Synthetic constructs encoding the optimised gene and a virus-derived wild type equivalent were separately cloned into a mammalian expression vector. CHO cells, transiently transfected with the optimised and wildtype NS1 vectors respectively, were sampled daily for 7 days, with the NS1 levels analysed in both the supernatant and cell fraction using Capture ELISA and Western blot techniques.
Transfection of the optimised NS1 sequence, successfully increased production of recombinant DENV and YFV NS1, with peak yields of up to 20 ug/ml detected in the supernatant of optimised DENV construct transfected cells at day 4-6 post-transfection. The increased protein yield achieved by codon optimisation falls within the biologically relevant range of 1-20 ug/ml, which therefore allows for a more streamlined pathway to NS1 protein characterisation and functional analysis by eliminating the need for downstream processing and possible endotoxin contamination.