Improvement of the transient expression system for production of recombinant proteins in plants

Tsuyoshi Yamamoto,Kentaro Ezura,Hugh S Mason,Hiroshi Ezura,Kenji Miura,Risa Okazawa,Ken Hoshikawa,Miyo Takaoka,Satoshi Fujita

doi:10.1038/s41598-018-23024-y

Tsuyoshi Yamamoto, Kentaro Ezura + Show 7 more

Open Access

https://doi.org/10.1038/s41598-018-23024-y

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Journal: Scientific Reports	Publication Date: Mar 19, 2018
Citations: 130	License type: open-access

Affiliation: University of Tsukuba, Arizona State University

Abstract

An efficient and high yielding expression system is required to produce recombinant proteins. Furthermore, the transient expression system can be used to identify the localization of proteins in plant cells. In this study, we demonstrated that combination of a geminiviral replication and a double terminator dramatically enhanced the transient protein expression level in plants. The GFP protein was expressed transiently in lettuce, Nicotiana benthamiana, tomatoes, eggplants, hot peppers, melons, and orchids with agroinfiltration. Compared to a single terminator, a double terminator enhanced the expression level. A heat shock protein terminator combined with an extensin terminator resulted in the highest protein expression. Transiently expressed GFP was confirmed by immunoblot analysis with anti-GFP antibodies. Quantitative analysis revealed that the geminiviral vector with a double terminator resulted in the expression of at least 3.7 mg/g fresh weight of GFP in Nicotiana benthamiana, approximately 2-fold that of the geminiviral vector with a single terminator. These results indicated that combination of the geminiviral replication and a double terminator is a useful tool for transient expression of the gene of interest in plant cells.

Highlights

Transgenic plants are generally used to obtain recombinant proteins or identify the localization of proteins
In the pBYR2HS vector, the alcohol dehydrogenase (AtADH) 5′-UTR region was replaced with tobacco mosaic virus (TMV) Ω and the heat shock protein (HSP) terminator was inserted into pBYR2fp, resulting in a double terminator construct (Fig. 1)
Each vector was transformed into Agrobacterium tumefaciens GV3101 and green fluorescent protein (GFP) was transiently expressed in N. benthamiana (Fig. 2A), lettuce Lactuca sativa (Fig. 2B), eggplants Solanum melongena (Fig. 2C), tomato Solanum lycopersicum fruits (Fig. 2D), tomato leaves (Fig. 2E), hot peppers Capsicum frutescens (Fig. 2F), melons Cucumis melo (Fig. 2G), orchids Phalaenopsis aphrodite (Fig. 2H), and roses (Fig. 2I)

Summary

Introduction

Transgenic plants are generally used to obtain recombinant proteins or identify the localization of proteins. Bean yellow dwarf virus (BeYDV), a Mastrevirus of the Geminiviridae family, contains a single-stranded circular DNA genome and uses a rolling circle mechanism to replicate its genome, resulting in a very high yield of copies This mechanism has been used for boosting protein expression in transgenic plants[6] and for transient expression of foreign proteins with efficiency in N. benthamiana leaves[7] and in lettuce leaves[5]. When the HSP and Ext terminators were used as a double terminator, the expression level was the highest and reached approximately 3.7 mg/g fresh weight (FW) in N. benthamiana This system could be applicable to N. benthamiana, and tomatoes, eggplants, lettuce, hot peppers, melons, and orchids. These results indicated that this system is a useful tool, with which to express specific proteins in plant cells

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Conclusion