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Expression and Self Assembly of Cowpea Chlorotic Mottle Virus Capsid Proteins in Pichia pastoris and Encapsulation of Fluorescent Myoglobin

  • Yuanzheng Wu (a1) (a2), Hetong Yang (a2) and Hyun-Jae Shin (a1)
Abstract:

Cowpea chlorotic mottle virus (CCMV) has been a model system for virus studies for over 40 years and now is considered to be a perfect candidate as nanoplatform for applications in materials science and medicine. The ability of CCMV to self assemble in vitro into virus-like particles (VLPs) or capsids makes an ideal reaction vessel for nanomaterial synthesis and entrapment. Here we report expression of codon optimized CCMV coat protein in Pichia pastoris and production of self assembled CCMV VLPs by large-scale fermentation. CCMV coat protein gene (573 bp) was synthesized according to codon preference of P. pastoris and cloned into pPICZA vector. The recombinant plasmid pPICZA-CP was transformed into P. pastoris GS115 by electroporation. The resulting yeast colonies were screened by PCR and analyzed for protein expression by SDS-PAGE. After large-scale fermentation CCMV coat protein yields reached 4.8 g L−1. The CCMV VLPs were purified by modified PEG precipitation followed by cesium chloride density gradient ultracentrifugation, and then analyzed by size exclusion fast performance liquid chromatography (FPLC), UV spectrometry and transmission electron microscopy. Myoglobin was used as a model protein to be encapsulated in CCMV VLPs. The fluorescence spectroscopy showed that inclusion of myoglobin had occurred. The results indicated the production of CCMV capsids by P. pastoris fermentation now available for utilization in pharmacology or nanotechnology fields.

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