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




Bipartite and tripartite Cucumber mosaic virus-based vectors for producing the Acidothermus cellulolyticus endo-1,4--glucanase and other proteins in non-transgenic plants - Download this document for free, or read online. Document in PDF available to download.

BMC Biotechnology

, 12:66

Plant biotechnology

Abstract

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--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--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~11g-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 ~21g-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.

KeywordsCucumber mosaic virusProtein productionEndoglucanaseAgrobacterium tumefaciencesViral vectorTransient protein expressionNicotiana benthamianaAbbreviationsCMVCucumber mosaic virus

GFPGreen fluorescent protein

CPCoat protein

ORFOpen reading frame

MPMovement protein

FWFresh weight

TSPTotal soluble protein

RFPRed fluorescent protein.

Electronic supplementary materialThe online version of this article doi:10.1186-1472-6750-12-66 contains supplementary material, which is available to authorized users.

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Author: MinSookHwang - BenjaminELindenmuth - KarenAMcDonald - BryceWFalk

Source: https://link.springer.com/







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