Expression and self-assembly of Heterocapsa circularisquama RNA virus-like particles synthesized in Pichia pastoris |
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Authors: | YuanZheng Wu Wonduck Kim Si-Wouk Kim Chi-Yong Eom HeTong Yang Hyun-Jae Shin |
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Institution: | 1. Department of Chemical and Biochemical Engineering, Chosun University, Gwangju, 501-759, Republic of Korea 4. Biotechnology Center of Shandong Academy of Sciences, Jinan, 250014, China 2. Department of Environmental Engineering, Chosun University, Gwangju, 501-759, Republic of Korea 3. Seoul Center, Korea Basic Science Institute, Seoul, 136-713, Republic of Korea
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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. |
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Keywords: | Heterocapsa circularisquama RNA virus(HcRNAV) virus-like particles(VLPs) expression self-assembly Pichia pastoris |
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