Optimization of expression and purification of recombinant S1 domain of the porcine epidemic diarrhea virus spike (PEDV- S1) protein inEscherichia coli

Abstract

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.

Cloning and Optimizing the Culture Parameters for Expression of R1 and R2 Repeat Regions of P97 Adhesin from Mycoplasma hyopneumoniae in Escherichia coli

“…Previous studies have reported that after adding the IPTG into the medium, the target protein begins to be synthesized and post-induction incubation time is necessary for recombinant protein production. The duration of post-induction incubation affects the overall folding, accumulation, and productivity of recombinant proteins in E. coli (Wong et al, 1998), and is affected by several factors such as strength of promotor, inducer concentration, solubility, and intrinsic properties of recombinant proteins (Noi and Chung, 2017). Our results suggest that the optimal post-induction incubation time for the highest expression of recombinant P97R1R2 protein in E. coli BL21(DE3) cells is 6 h.…”

NosZ gene cloning, reduction performance and structure of Pseudomonas citronellolis WXP-4 nitrous oxide reductase

“…10,40 The addition of the inducer during the lag phase makes bacterial growth difficult; its addition during the stabilization phase inhibits the expression of the target gene. 41 Generally, the optimal OD 600 interval for gene induced expression should be in the range of 0.5-0.8. In this study, the OD 600 was selected as 0.6, and the culture time was 2 h. The optimal induce conditions were 1.0 mM IPTG, 5 h, and 30 C. Aer protein extraction, isolation, and purication, the expression of the nosZ gene was examined by SDS-PAGE gel electrophoresis, and the results were shown in Fig.…”

Oral Administration of Coronavirus Spike Protein Provides Protection to Newborn Pigs When Challenged with PEDV

“…These include immunogens based on the S1 moiety [11], the S2 moiety [12], and a smaller portion known as the core neutralizing epitope or COE (amino acids 499-638) that has been identified as containing neutralizing epitopes [13]. However, the prototype vaccines require the purification of the S protein, which has been difficult to produce at high levels in several recombinant systems [11,14,15].…”