Generation of soluble, disulfide-rich JEV NS1 protein recognizable by anti-NS1 antibodies through a simplified, in vitro refolding approach

Co-existence of Japanese Encephalitis virus (JEV) with highly homologous antigenic epitopes results in antibodybased serodiagnosis being inaccurate at detecting and distinguishing JEV from other flaviviruses. This often causes misdiagnosis and inefficient treatments of flavivirus infection. Generation of JEV NS1 protein remains a challenge as it is notably expressed in the form of inactive aggregates known as inclusion bodies using bacterial expression systems. This study evaluated two trxB and gor E. coli strains in producing soluble JEV NS1 via a coldshock expression system. High yield of JEV NS1 inclusion bodies was produced using cold-shocked expression system. Subsequently, a simplified yet successful approach in generating soluble, active JEV NS1 protein through solubilization, purification and in vitro refolding of JEV NS1 protein from inclusion bodies was developed. A stepwise dialysis refolding approach was used to facilitate JEV NS1 refolding. The authenticity of the refolded JEV NS1 was confirmed by specific antibody binding on indirect ELISA commercial anti-NS1 antibodies which showed that the refolded JEV NS1 was highly immunoreactive. This presented approach is cost-effective, and negates the need for mammalian or insect cell expression systems in order to synthesize this JEV NS1 protein of important diagnostic and therapeutic relevance in Japanese Encephalitis disease.

Automated summary

The co-existence of JEV with highly homologous antigenic epitopes poses challenges in accurate serodiagnosis, leading to misdiagnosis and ineffective treatments. This study successfully generated soluble and active JEV NS1 protein through a cold-shocked E. coli expression system, providing a cost-effective alternative to mammalian or insect cell expression systems for JEV NS1 synthesis.