Abstract
Heterocapsa circularisquama RNA virus (HcRNAV) is the first single-stranded RNA virus to be characterized that infects dinoflagellates. The ability of HcRNAV coat protein (HcRNAV CP) to self-assemble into virus-like particles (VLPs) in vitro suggested that heterologous expression was possible, and that the VLPs might be ideal nanocontainers for the targeted delivery of genes and chemicals. In this paper, we report the expression of a codon-optimized HcRNAV 109 CP gene in Pichia pastoris and the production of self-assembled HcRNAV VLPs using large-scale fermentation. The HcRNAV 109 CP gene was synthesized according to the codon preference of P. pastoris and cloned into a pPICZA vector. The recombinant plasmid pPICZA-CPsyns was transformed into P. pastoris by electroporation. The resulting yeast colonies were screened by PCR and analyzed for protein expression by SDS polyacrylamide gel electrophoresis. After large-scale fermentation, the yield of HcRNAV CPsyns reached approximately 2.5 g L−1 within 4 d. The HcRNAV VLPs were purified using PEG precipitation followed by cesium chloride density gradient ultracentrifugation, and were subsequently analyzed using UV spectrophotometry and transmission electron microscopy. Fluorescence dye-labeled myoglobin was loaded into the cages of the HcRNAV VLPs and the encapsulation was confirmed by fluorescence spectroscopy. The results point to the possible utilization in pharmacology or nanotechnology of HcRNAV VLPs produced by P. pastoris fermentation.
Highlights
Harmful algal blooms (HABs), known as red tides, have had a negative impact on aquatic ecosystems and have increasingly become a threat to human and marine health [1,2]
We report on the heterologous synthesis of Heterocapsa circularisquama RNA virus (HcRNAV) 109 coat protein by P. pastoris
To enhance the efficiency of gene expression, the HcRNAV CPsyns gene was optimized to the codon usage of P. pastoris (Figure 1)
Summary
Harmful algal blooms (HABs), known as red tides, have had a negative impact on aquatic ecosystems and have increasingly become a threat to human and marine health [1,2]. The toxic or harmful phytoplanktons that cause HABs are commonly dinoflagellates, such as Alexandrium and Karenia [8]. Many efforts have been made to control harmful algal blooms with little success [9]. Viruses that are abundant in marine systems replicate rapidly and tend to be host-specific, suggesting that single algal species. Parasites have the potential to control algal bloom species, but their specific role in this regard is largely unknown [13]. Heterocapsa circularisquama RNA virus (HcRNAV) is the first single-stranded RNA virus to be characterized that infects dinoflagellates [14]. The virus clones have been divided into types CY and UA (HcRNAV109 and HcRNAV34, respectively), based on their host strain specificity [17]
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