Classification of cryo electron microscopy images, noisy tomographic images recorded with unknown projection directions, by simultaneously estimating reconstructions and application to an assembly mutant of Cowpea Chlorotic Mottle Virus and portals of the bacteriophage P22

2010 ◽  
Author(s):  
Junghoon Lee ◽  
Yili Zheng ◽  
Zhye Yin ◽  
Peter C. Doerschuk ◽  
John E. Johnson
Keyword(s):  
Electron Microscopy ◽  
Mottle Virus ◽  
Bacteriophage P22 ◽  
Cowpea Chlorotic Mottle Virus ◽  
Tomographic Images ◽  
Cryo Electron Microscopy ◽  
Chlorotic Mottle Virus ◽  
Chlorotic Mottle ◽  
Microscopy Images

Expression and Self Assembly of Cowpea Chlorotic Mottle Virus Capsid Proteins inPichia pastorisand Encapsulation of Fluorescent Myoglobin

2011 ◽  
Vol 1317 ◽  
Author(s):  
Yuanzheng Wu ◽  
Hetong Yang ◽  
Hyun-Jae Shin
Keyword(s):  
Coat Protein ◽  
Large Scale ◽  
Materials Science ◽  
Cesium Chloride ◽  
Size Exclusion ◽  
Mottle Virus ◽  
Cowpea Chlorotic Mottle Virus ◽  
Chlorotic Mottle Virus ◽  
Chlorotic Mottle

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 assemblein vitrointo 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 inPichia pastorisand production of self assembled CCMV VLPs by large-scale fermentation. CCMV coat protein gene (573 bp) was synthesized according to codon preference ofP. pastorisand cloned into pPICZA vector. The recombinant plasmid pPICZA-CP was transformed intoP. pastorisGS115 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 byP. pastorisfermentation now available for utilization in pharmacology or nanotechnology fields.

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