The PEDV-S1 protein, one of the members of a large family of PEDV, is significantly upregulated and has been targeted as a biomarker of cellular stress in several studies. Herein, conditions were optimized to increase the yield of recombinant PEDV-S1 protein in its native state, primarily focusing on the optimization of upstream processing parameters that lead to an increase in the specific as well as volumetric yield of the protein. The results showed that the production of PEDV-S1 was increased proportionally with increased incubation temperature up to 37 C. Induction with 10 lM IPTG was sufficient to induce the expression of PEDV-S1 which was 100 times less than the normally used IPTG concentration. Furthermore, the results indicate that induction during early to late exponential phase produced relatively high levels of PEDV-S1 in soluble form. In addition, 5 h of post-induction incubation was found to be optimal to produce folded PEDV-S1 with higher specific and volumetric yield. Subsequently, highly pure and homogenous PEDV-S1 preparation was obtained using metal affinity and size exclusion chromatography. Taken together, the results showed successful production of electrophoretically pure recombinant PEDV-S1 protein in Escherichia coli (E. coli) in milligram quantities from shake flask liquid culture.