Transient protein expression systems in plants and their applications

Abstract

The production of recombinant proteins is important in academic research to identify protein functions. Moreover, recombinant enzymes are used in the food and chemical industries, and high-quality proteins are required for diagnostic, therapeutic, and pharmaceutical applications. Though many recombinant proteins are produced by microbial or mammalian cell-based expression systems, plants have been promoted as alternative, cost-effective, scalable, safe, and sustainable expression systems. The development and improvement of transient expression systems have significantly reduced the period of protein production and increased the yield of recombinant proteins in plants. In this review, we consider the importance of plant-based expression systems for recombinant protein production and as genetic engineering tools.

Keywords: Tsukuba system; ascorbic acid; deconstructed vector; necrosis; transient expression.

Figure 1. Schematic representation of the T-DNA regions of plasmids pBYR2HS, pTKB2, and pTKB3. pBYR2HS is a previously produced vector (Yamamoto et al. 2018a). 35S-p×2, CaMV 35S promoter with double-enhanced element; AtADH5′, 5′-untranslated region (UTR) of Arabidopsis thaliana alcohol dehydrogenase gene; TMV Ω, 5′-leader sequence of the tobacco mosaic virus; HSPter, terminator of a heat shock protein gene; Ext3′, tobacco extensin gene 3′ element; 35Ster, terminator of CaMV 35S; NOSter, NOS terminator; LIR, long intergenic region of the bean yellow dwarf virus (BeYDV) genome; SIR, short intergenic region of the BeYDV genome; C1/C2, BeYDV ORFs C1 and C2 encoding for replication initiation protein (Rep) and RepA, respectively; LB and RB, the left and right borders of the T-DNA region, respectively; Nos-p and Nos-t, NOS promoter and terminator, respectively; p19, a gene-silencing suppressor gene from the tomato bushy stunt virus; GOI, gene of interest.

Figure 2. Agrobacterium culture conditions and concentrations for achieving efficient agroinfiltration and protein expression levels in N. benthamiana. (A) Agrobacterium carrying pBYR2HS-EGFP was agroinfiltrated (with 1-ml syringes) at different OD₆₀₀ into N. benthamiana. GFP emission was observed 3 days post agroinfiltration (dpa) with an ultraviolet-absorbing filter, Fujifilm SC-52. (B) Agrobacterium carrying pBYR2HS-EGFP was resuspended either in agroinfiltration MES buffer (10 mM MES, pH 5.6, 10 mM MgCl₂, 100 μM acetosyringone; MES) or Phosphate buffer (10 mM potassium phosphate, pH 5.6, 10 mM MgCl₂, 100 μM acetosyringone; P buf.) at OD₆₀₀=0.5, and used to agroinfiltrate N. benthamiana. GFP emission was observed with an ultraviolet-absorbing filter, Fujifilm SC-52, 3 dpa. (C) Agrobacterium resuspensions containing either pBYR2HS-EGFP, pTKB2-EGFP, or pTKB3-EGFP were adjusted to OD₆₀₀=0.5 or 0.1 and agroinfiltrated into N. benthamiana. GFP emission was detected at 3 dpa. (D) Total soluble proteins, which were extracted from 0.2 mg FW N. benthamiana leaves agroinfiltrated with Agrobacterium carrying pBYR2HS-EGFP, pTKB2-EGFP, or pTKB3-EGFP, were separated by SDS-PAGE, and stained with Coomassie Brilliant Blue (CBB) dye. (E) The relative amount of GFP was calculated based on protein band intensities in CBB-stained gels using ImageJ software. Data represent means±SE (n=4–6).

Figure 3. Necrosis of leaves is suppressed by foliar spraying of high concentrations of sodium ascorbate (AsA). After expressing either human cullin protein, hCul1, or F-box protein hFbxw7 in agroinfiltrated leaves, tissue necrosis (gray area) was observed (A), but not when leaves were sprayed with 200 mM AsA (B). AsA was applied by foliar spray (C).