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.

SARS‐CoV‐2 spike trimer vaccine expressed in Nicotiana benthamiana adjuvanted with Alum elicits protective immune responses in mice

“…Additionally, five histidine residues (His5) and an HDEL motif (used as a purification tag and ER retention signal, respectively) were added to the C terminus of the FD or mCor1 to yield the final constructs BiP:S Δ (TMD‐C):FD:His5:HDEL (S ft ) and BiP:S Δ (TMD‐C):mCor1:His5:HDEL (S ct ), respectively. First, we produced these proteins in N. benthamiana using the transient expression method (Song et al ., 2022 ).…”

Production of active Exendin-4 in Nicotiana benthamiana and its application in treatment of type-2 diabetics

“…Thus, we fused to Exdn-4 a few domains that were intended for solubilization of recombinant protein, ER targeting, affinity tagging for purification, and also translational enhancement. First, we utilized the GB1 domain, which is known to increase the expression level of its fusion proteins several-fold ( Song et al., 2022 ). In addition, we intended to accumulate proteins in the ER by targeting recombinant proteins to the ER using the signal peptide of Arabidopsis BiP1, including seven amino acids after the signal peptide cleavage site to favor proper peptide removal.…”

“…Finally, an enterokinase site (DDDDK) and the Exdn-4 domain were fused to the C-terminus of ddCBDm to give BiP : GB1:ddCBDm:8xHis : EK:Exendin-4 ( BGC : Exdn-4 for short) ( Figure 1A ). The recombinant gene was under the control of MacT promoter ( Song et al., 2022 ). It also contained the gene silencing suppressor P38 of turnip crinkle virus coat protein, so there was no need to provide P38 in a separate vector.…”

“…A DNA fragment of 2L:EK : Exdn-4 consisting of 2 copies of GGGS as a linker, an enterokinase site, and Exendin-4 was chemically synthesized (Gene Universal, USA). 2L:EK : Exdn-4 was digested with XmaI and XhoI restriction endonucleases and ligated into the pTEX1::BiP : GB1:HL:GFP vector ( Song et al., 2022 ), digested with the same endonucleases to give BiP : GB1:HL:2L:EK : Exdn-4 . Subsequently, a DNA fragment, ddCBDm with 8xHis, that had been chemically synthesized (Gene Universal, USA) was digested with BamH1 and XmaI restriction endonucleases and ligated into pTEX1::BiP : GB1:HL:2L:EK : Exdn-4 digested with BamH1 and XmaI restriction endonucleases to give pTEX1::B:G:ddCBDm : Exdn-4 ( BGC : Exdn-4 ).…”

“…The two constructs BGC : Exdn-4 and BGC : Exdn-4:HDEL together with GFP were transiently expressed in N. benthamiana via Agrobacterium -mediated infiltration ( Song et al., 2022 ). Total leaf tissues were harvested and homogenized using an active Motif Dounce Homogenizer (Thermofisher Catalog No.NC0569256) in 2 mL of homogenization buffer (50 mM Tris-HCl, pH 8.2, 20% [v/v] glycerol, 1 mM DTT, 1x protease inhibitor cocktail) per gram of tissue.…”