Bipartite and tripartite Cucumber mosaic virus-based vectors for producing the Acidothermus cellulolyticus endo-1,4-β-glucanase and other proteins in non-transgenic plants

BMC Biotechnology

Research Article

Karen A McDonald¹  Benjamin E Lindenmuth²  Bryce W Falk³  Min Sook Hwang³ 

[1] Department of Chemical Engineering and Materials Science, University of California, One Shields Avenue, 95616, Davis, CA, USA;Department of Chemical Engineering and Materials Science, University of California, One Shields Avenue, 95616, Davis, CA, USA;Bayer HealthCare Pharmaceuticals, 800 Dwight Way, 94710, Berkeley, CA, USA;Department of Plant Pathology, University of California, One Shields Avenue, 95616, Davis, CA, USA;

关键词: Cucumber mosaic virus;    Protein production;    Endoglucanase;    Agrobacterium tumefaciences;    Viral vector;    Transient protein expression;    Nicotiana benthamiana;   

DOI  :  10.1186/1472-6750-12-66

 received in 2012-06-14, accepted in 2012-09-11,  发布年份 2012

来源: Springer

【 摘 要 】

BackgroundUsing plant viruses to produce desirable proteins in plants allows for using non-transgenic plant hosts and if necessary, the ability to make rapid changes in the virus construct for increased or modified protein product yields. The objective of this work was the development of advanced CMV-based protein production systems to produce Acidothermus cellulolyticus endo-1, 4-β-glucanase (E1) in non-transgenic plants.ResultsWe used two new Cucumber mosaic virus (CMV)-based vector systems for producing the green fluorescent protein (GFP) and more importantly, the Acidothermus cellulolyticus endo-1, 4-β-glucanase (E1) in non-transgenic Nicotiana benthamiana plants. These are the inducible CMVin (CMV-based inducible) and the autonomously replicating CMVar (CMV-based advanced replicating) systems. We modified a binary plasmid containing the complete CMV RNA 3 cDNA to facilitate insertion of desired sequences, and to give modifications of the subgenomic mRNA 4 leader sequence yielding several variants. Quantitative RT-PCR and immunoblot analysis showed good levels of CMV RNA and coat protein accumulation for some variants of both CMVin and CMVar. When genes for E1 or GFP were inserted in place of the CMV coat protein, both were produced in plants as shown by fluorescence (GFP) and immunoblot analysis. Enzymatic activity assays showed that active E1 was produced in plants with yields up to ~ 11 μg/g fresh weight (FW) for specific variant constructs. We also compared in vitro CMV genomic RNA reassortants, and CMV RNA 3 mutants which lacked the C’ terminal 33 amino acids of the 3A movement protein in attempts to further increase E1 yield. Taken together specific variant constructs yielded up to ~21 μg/g FW of E1 in non-transgenic plants.ConclusionsIntact, active E1 was rapidly produced in non-transgenic plants by using agroinfiltration with the CMV-based systems. This reduces the time and cost compared to that required to generate transgenic plants and still gives the comparable yields of active E1. Our modifications described here, including manipulating cloning sites for foreign gene introduction, enhance the ease of use. Also, N. benthamiana, which is particularly suitable for agroinfiltration, is a very good plant for transient protein production.

【 授权许可】

Unknown   
© Hwang et al.; licensee BioMed Central Ltd. 2012. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.