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Expression and Self Assembly of Cowpea Chlorotic Mottle Virus Capsid Proteinsin Pichia pastoris and Encapsulation of FluorescentMyoglobin

Published online by Cambridge University Press:  28 January 2011

Yuanzheng Wu ,
Hetong Yang  and
Hyun-Jae Shin
Yuanzheng Wu
Affiliation:
Department of Chemical and Biochemical Engineering, Chosun University, #375 Seosuk-dong, Dong-gu, Gwangju, 501-759, Republic of Korea Biotechnology Center of Shandong Academy of Sciences, No. 19 Keyuan Road, Jinan, 250014, China, P. R.
Hetong Yang
Affiliation:
Biotechnology Center of Shandong Academy of Sciences, No. 19 Keyuan Road, Jinan, 250014, China, P. R.
Hyun-Jae Shin
Affiliation:
Department of Chemical and Biochemical Engineering, Chosun University, #375 Seosuk-dong, Dong-gu, Gwangju, 501-759, Republic of Korea
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Abstract:

Cowpea chlorotic mottle virus (CCMV) has been a model system for virus studies for over40 years and now is considered to be a perfect candidate as nanoplatform for applicationsin materials science and medicine. The ability of CCMV to self assemble invitro into virus-like particles (VLPs) or capsids makes an ideal reactionvessel for nanomaterial synthesis and entrapment. Here we report expression of codonoptimized CCMV coat protein in Pichia pastoris and production of selfassembled CCMV VLPs by large-scale fermentation. CCMV coat protein gene (573 bp) wassynthesized according to codon preference of P. pastoris and cloned intopPICZA vector. The recombinant plasmid pPICZA-CP was transformed into P.pastoris GS115 by electroporation. The resulting yeast colonies were screenedby PCR and analyzed for protein expression by SDS-PAGE. After large-scale fermentationCCMV coat protein yields reached 4.8 g L−1. The CCMV VLPs were purified bymodified PEG precipitation followed by cesium chloride density gradientultracentrifugation, and then analyzed by size exclusion fast performance liquidchromatography (FPLC), UV spectrometry and transmission electron microscopy. Myoglobin wasused as a model protein to be encapsulated in CCMV VLPs. The fluorescence spectroscopyshowed that inclusion of myoglobin had occurred. The results indicated the production ofCCMV capsids by P. pastoris fermentation now available for utilization inpharmacology or nanotechnology fields.

Keywords

biological synthesis (assembly)nanostructuretransmission electron microscopy (TEM)

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Type
Articles
Information
MRS Online Proceedings Library (OPL) , Volume 1317: Symposium RR – Interdisciplinary Approaches to Safe Nanotechnologies , 2011 , mrsf10-1317-rr03-05
Copyright
Copyright © Materials Research Society 2011

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