Seed-Specific Expression of Spider Silk Protein Multimers Causes Long-Term Stability

Abstract

Seeds enable plants to germinate and to grow in situations of limited availability of nutrients. The stable storage of different seed proteins is a remarkable presumption for successful germination and growth. These strategies have been adapted and used in several molecular farming projects. In this study, we explore the benefits of seed-based expression to produce the high molecular weight spider silk protein FLAG using intein-based trans-splicing. Multimers larger than 460 kDa in size are routinely produced, which is above the native size of the FLAG protein. The storage of seeds for 8 weeks and 1 year at an ambient temperature of 15°C does not influence the accumulation level. Even the extended storage time does not influence the typical pattern of multimerized bands. These results show that seeds are the method of choice for stable accumulation of products of complex transgenes and have the capability for long-term storage at moderate conditions, an important feature for the development of suitable downstream processes.

Keywords: intein; protein trans-splicing; seed expression; spider silk proteins; tobacco.

FIGURE 1

Expression of synthetic FLAG multimers in tobacco seeds. (A) Schematic representation of the plant expression cassette for seed-specific expression of intein-based assembled high molecular weight synthetic FLAG. Abbreviations: USP, unknown seed protein promoter; SP, legumin B4 signal peptide; KDEL, ER retention sequence; IntC/IntN, C- and N-terminal intein sequences of the Synechocystis sp. DnaB; GS-linker, flexible (GGGGS)₃ spacer; c-myc, immunodetection tag. (B) Extracts of T₁ seeds of four different transformed tobacco plants expressing the FLAG-Intein-c-myc protein (USP-FIC) and of corresponding wild type cultivar Nicotiana tabacum cv. Samsun NN were separated by gradient SDS-PAGE (4–10% PAA) with 30 μg total soluble seed protein loaded per lane reflecting 4.1 seeds (USP-FIC 28), 4.0 seeds (USP-FIC 14), 4.7 seeds (USP-FIC 10), and 3.9 seeds (USP-FIC 5). FLAG multimers were immunodetected by Western blotting based on the c-myc tag. c-myc standards (a) 0.5 ng, (b) 1 ng, (c) 2 ng of anti-TNF-V_HH-100xELP (Conrad et al., 2011); WT, wild type.

FIGURE 2

Accumulation of FLAG multimers in seeds of two different tobacco varieties. (A) Analysis of ripe T₃ seeds of N. tabacum cv. SNN USP-FIC line 28/10 and T₂ seeds of N. tabacum cv. Petit Havana USP-FIC line 49 by gradient SDS-PAGE (4–10% PAA) with 20 μg total soluble seed protein per lane, Western blotting and immunodetection based on the c-myc tag; kDa, kilodalton. (B) Transgenic USP-FIC line 49 with a genomic background of N. tabacum cv. Petit Havana as well as the corresponding wild type plants showed a faster vegetative growth and flowered 8 days earlier than Samsun NN-genome-based FLAG overexpressing plants of USP-FIC line 28/10 and its corresponding wild type plants.

FIGURE 3

Intein-mediated formation of high molecular weight FLAG proteins in ripening tobacco seeds. FLAG precursor proteins under the control of the USP promoter were analyzed in developing tobacco seeds at 18 DAF (1), 21 DAF (2) and in ripe tobacco seeds (3). Extracts from 31.2 seeds were loaded per lane. Seed extracts were separated by gradient SDS-PAGE (4–10% PAA), and FLAG proteins were visualized by immunodetection based on the c-myc tag. DAF, days after flowering; kDa, kilodalton; WT, wild type.

FIGURE 4

Stability analysis of multimeric FLAG proteins in transgenic tobacco seeds. Accumulation of high molecular weight FLAG multimers from T₃ seeds of USP-FIC 28/10/11 (cv. Samsun NN) stored for 8 weeks at 15°C and 49% humidity (A) and T₂ seeds of USP-FIC 49/9 (cv. Petit Havana) stored under identical conditions. (B) Seed extracts were separated by gradient SDS-PAGE (4–10% PAA). Line USP-FIC 28/10/11: 1.4 seeds per lane, (2) 2.8 seeds per lane, (3) 5.6 seeds per lane; Line USP-FIC 49/9: (1) 1.7 seeds per lane, (2) 3.4 seeds per lane, (3) 6.8 seeds per lane. c-myc immunoblot standards: (4) 0.1 ng, (5) 0.25 ng (6) 0.5 ng, (7) 1.0 ng, (8) 2.0 ng of anti-TNF-V_HH-100xELP. (C) Stability of FLAG multimers in the T₁ seeds of transformed T₀ plant USP-FIC 28 after 1 year of storage at 15°C and 49% humidity compared to the FLAG accumulation pattern in seed extracts of freshly harvested T₂ seeds of selected heterozygous USP-FIC 28/plants. Seed extracts were separated by gradient SDS-PAGE (3–10% PAA) with 20 μg total soluble seed protein per lane and visualized by immunodetection based on the c-myc tag. kDa, kilodalton.

FIGURE 5

Analysis of endogenous seed proteins in transgenic tobacco seeds. Extracts from ripe seeds of two transgenic lines (USP-FIC 28/10/15 and USP-FIC 49/9) and their corresponding wild type cultivars were prepared. Total amounts of 5 μg total soluble protein were separated by SDS-PAGE (12% PAA) and visualized by Coomassie staining.