Recombinant expression of Zika virus NS1 protein in Escherichia coli: effects of fusion strategy and culture conditions.
Zika virus (ZIKV) nonstructural protein 1 (NS1) represents a valuable diagnostic target due to its early secretion during infection and reduced cross-reactivity with other flaviviruses. Optimization of NS1 expression conditions is essential for obtaining large-scale protein production. The use of bacterial expression systems as Escherichia coli offers a cost-effective platform. We developed two molecular cloning strategies to express full-length ZIKV NS1 (1053 bp) from Cuban strain 9661/2016 using pQE30/XL1-Blue and pET50b(+)/BL21(DE3) expression systems. The ns1 gene cloned via pGEM-T intermediate vector, and subcloned into expression vectors by using directional restriction-based strategy. The effects of culture medium and IPTG concentration on bacterial growth and protein accumulation was evaluated in order to optimizing the NS1 expression conditions. Both expression systems successfully produced recombinant ZIKV NS1. However, the BL21(DE3)/pET50b(+) system yielded soluble NS1, whereas the XL1-Blue/pQE30 system predominantly produced insoluble protein. Culture medium exerted a greater effect on protein expression than IPTG concentration, with LB medium and 0.1 mM IPTG providing the most favorable conditions for recombinant NS1 production. Notably, XL1-Blue demonstrated competency for protein expression. The pET50b(+)-ns1/BL21(DE3) system offers decisive advantages through soluble expression, eliminating refolding requirements for downstream purification process. This work establishes optimized protocols for ZIKV NS1 expression applicable to obtaining a recombinant protein for developing an ELISA system and broadly transferable to other flavivirus antigens.