The B1 Domain of Streptococcal Protein G Serves as a Multi-Functional Tag for Recombinant Protein Production in Plants

Abstract

Plants have long been considered a cost-effective platform for recombinant production. A recently recognized additional advantage includes the low risk of contamination of human pathogens, such as viruses and bacterial endotoxins. Indeed, a great advance has been made in developing plants as a "factory" to produce recombinant proteins to use for biopharmaceutical purposes. However, there is still a need to develop new tools for recombinant protein production in plants. In this study, we provide data showing that the B1 domain of Streptococcal protein G (GB1) can be a multi-functional domain of recombinant proteins in plants. N-terminal fusion of the GB1 domain increased the expression level of various target proteins ranging from 1.3- to 3.1-fold at the protein level depending on the target proteins. GB1 fusion led to the stabilization of the fusion proteins. Furthermore, the direct detection of GB1-fusion proteins by the secondary anti-IgG antibody eliminated the use of the primary antibody for western blot analysis. Based on these data, we propose that the small GB1 domain can be used as a versatile tag for recombinant protein production in plants.

Keywords: GB1; Nicotiana benthamiana; biopharmaceutical proteins; plant-based molecular pharming; protein folding.

FIGURE 1

GB1 dramatically increases the expression level of GFP in the leaves of Nicotiana benthamiana. (A–C) Schematic representation of constructs. BiP-EK-GFP-HDEL and BiP-GB1- EK-GFP-HDEL (A), RbcS_tp-EK-GFP-HDEL and RbcS_tp-GB1-EK-GFP-HDEL (B), and GFP-HDEL and GB1-EK-GFP-HDEL (C). BiP, the leader sequence of BiP; RbcS_tp, transit peptide of the rubisco complex small subunit. (D–F) Images of GFP fluorescence. GFP fluorescent signals of the constructs targeted to ER (D), chloroplasts (E), and cytosol (F) were measured from infiltrated leaves at 3 days post infiltration (dpi). (G–I) Coomassie brilliant blue (CBB)-stained GFP bands. Total protein extracts from infiltrated N. benthamiana leaves at 3, 5, and 7 dpi were separated by SDS-PAGE and stained with Coomassie brilliant blue. ER (G), chloroplast (H), and cytosol (I)-localized GFP (red asterisks) and GB1-GFP (blue asterisks). Band intensity was quantified and represented as a relative value to the GFP alone. Three independent experiments were carried out to quantify the signal intensity. Results in panels (G,I) are the mean ± SD (n = 3). Asterisks indicate a significant difference (Student’s t-test; one asterisk and three asterisks indicate P < 0.05 and P < 0.001, respectively).

FIGURE 2

C-terminal fusion of GB1 does not enhance the expression level of GFP in the ER of Nicotiana benthamiana. (A) Schematic representation of constructs. BiP-EK-GFP-HDEL (top), BiP-GB1-EK-GFP-HDEL (middle), and BiP-EK-GFP-GB1-HDEL (bottom). (B) Images of GFP fluorescence. The indicated constructs were expressed in leaf tissues of N. benthamiana via agroinfiltration, and images were taken at 3, 5, and 7 DPI. GFP (top), GB1-GFP (middle), and GFP-GB1 (bottom). (C) Quantification of GFP fluorescent signals. Signal intensity was quantified using three DPI samples. Three independent experiments were carried out. (D) GFP level by CBB staining. Total protein extracts from leaves harvested at 3, 5, and 7 DPI were separated by SDS-PAGE and stained with CBB. GFP band intensity was quantified from the CBB-stained gel after SDS-PAGE. (E) Quantification of GFP levels. The GFP bands in Figure (D) were quantified and represented as relative values. Results for panels (C,E) are the mean ± SD (n = 3). Asterisks indicate a significant difference (Student’s t-test; one, two, and three asterisks indicate P < 0.05, 0.001 < P < 0.05, and P < 0.001, respectively).

FIGURE 3

GB1 enhances the expression of various target proteins in Nicotiana benthamiana. (A–C) SDS-PAGE analysis of target protein levels. The indicated target proteins were transiently expressed in Nicotiana benthamiana. These proteins from 0.1 g infiltrated tissues for each sample were purified using Ni²⁺-NTA affinity column chromatography and separated by SDS-PAGE. The gels were stained with CBB. (A) Lanes 1–3, purified hIL6; lanes 4–6, GB1-hIL6. (B) Lanes 1–3, purified HA; lanes 4–6, GB1-HA. (C) Lanes 1–3, purified CTB; lanes 4–6, GB1-CTB. Red arrows indicate the target proteins. (D–F) Quantification of signal intensity. The signal intensity of target protein bands in Figures (A–C) was quantified and represented in panels (D–F), respectively, as relative values. Results in panels (D–F) are the mean ± SD (n = 3). Asterisks indicate a significant difference (Student’s t-test; two asterisks indicate 0.001 < P < 0.05). (G) SDS-PAGE (15%) analysis of GB1-CTB cleaved by TEV protease at 10 and 25^°C overnight.

FIGURE 4

GB1 fusion leads to an enhanced translation rate in vitro. LUC and GB1-LUC were transcribed and translated in vitro in wheat germ extracts. During the reaction, 5 μl were taken from the 50 μl reaction volume at 30, 60, and 120 min and diluted 24-fold. The RLUC activity was measured using the Renilla Luciferase Assay System. Three independent translation reactions were carried out. The error bar is the mean ± SD (n = 3). Asterisks indicate a significant difference (Student’s t-test; three asterisks, P < 0.001).

FIGURE 5

The β-hairpin structure of GB1 is critical for the thermal stability of CTB but has no contribution to the increase in protein expression. (A) Protein sequences of GB1 and GB1[W43A]. (B) The β-hairpin structure of GB1. (C) Images of GFP fluorescence. The indicated constructs were expressed in Nicotiana benthamiana leaves, and images were taken at three DPI. (D) Quantification of GFP signals. GFP signals in Figure (C) were quantified and represented as relative values. The error bar is the mean ± SD (n = 3). Single Asterisks indicate a significant difference (Student’s t-test, P < 0.05). (E) Thermal stability of GB1 and GB1[W43A]-fused CTB. The indicated constructs were expressed in N. benthamiana via Agrobacterium-mediated infiltration. Proteins were purified using Ni²⁺-NTA affinity column chromatography. Purified proteins (2 μg) were incubated at 60^oC for 1, 2, and 3 days and separated by SDS-PAGE. The gel was stained with CBB. Black asterisks indicate protein breakdown products.

FIGURE 6

GB1 is directly detected by IgG from various animals. (A) Protein sequences of GB1, GB1[E27A], and GB1[E27A/W43A]. Asterisks indicate mutations. (B) The 3-D structures of human Fc and GB1. These structures were obtained from the public database. Human Fc, PBD: 1aj7; GB1, PBD: 2gi9. The red color at the GB1 structure indicates E27, which functions as a knob for the hole of Fc (H433 and N434, red); the blue color at the GB1 structure indicates W43, which functions as a hole for the knob of Fc (I253 and S254, blue). (C) Strong binding of hFc to GB1 but not GB1 mutants. The indicated constructs were expressed in Nicotiana benthamiana, and total protein extracts were subjected to MCC beads-based protein pull-down. The proteins bound to the MCC beads were analyzed by western blotting using anti-CBM3 and anti-His antibodies. (D) Western blot analysis. Total protein extracts from N. benthamiana expressing the indicated constructs were analyzed by western blotting using the anti-His antibody. RbcL stained with CBB was used as a loading control. (E) Binding of various IgGs to GB1. Protein extracts used in Figure (D) were separated by SDS-PAGE and analyzed by western blotting using sheep anti-Mouse IgG (Sheep), goat anti-Human IgG (Goat), and mouse anti-Goat IgG (Mouse) without primary anti-His antibody. CTB, lane 1; GB1-CTB, lane 2; GB1[E27A]-CTB, lane 3; GB1[W43A]-CTB, lane 4. All constructs contain a His-tag at the C-terminus but are omitted to simplify the description.