Expression and Self Assembly of Cowpea Chlorotic Mottle Virus Capsid Proteins in Pichia pastoris and Encapsulation of Fl
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Expression and Self Assembly of Cowpea Chlorotic Mottle Virus Capsid Proteins in Pichia pastoris and Encapsulation of Fluorescent Myoglobin Yuanzheng Wu1,2, Hetong Yang2 and Hyun-Jae Shin1 1, Department of Chemical and Biochemical Engineering, Chosun University, #375 Seosukdong, Dong-gu, Gwangju, 501-759, Republic of Korea 2, Biotechnology Center of Shandong Academy of Sciences, No. 19 Keyuan Road, Jinan, 250014, China, P. R. 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. 1. Introduction Viruses and virus-like particle (VLPs) have recently been employed as potential building blocks for materials science and nanotechnology [1,2]. They typically consist of protein shells (termed as capsids) which surround a central core of DNA or RNA. The capsids are composed of identical coat protein (CP) subunits that permit manipulation by genetic and chemical modifications [3-5]. As nanoscale assemblies, viruses have sophisticated yet highly organized structural features. Therefore, viruses and VLPs offer a unique platform where functional motifs can be attached on their capsids precisely or encapsulated inside the cages, which is a big advantage over synthetic nanoparticles [6,7]. Cowpea chlorotic mottle virus (CCMV), a plant virus, has been a model system for virus studies since 1960s [8]. As a member of Bromoviradae family, CCMV is an icosahedral virus with external diameter of 28 nm and a well-defined inner cavity with a diameter of approximately 18 nm. The capsid comprises 90 copies of coat protein homodimers arranged with T=3 Caspar-Klug symmetry around a central RNA strand [9,10]. The 20 kDa CP subunit
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