In Vivo Removal of N-Terminal Fusion Domains From Recombinant Target Proteins Produced in Nicotiana benthamiana

Abstract

Plants show great potential for producing recombinant proteins in a cost-effective manner. Many strategies have therefore been employed to express high levels of recombinant proteins in plants. Although foreign domains are fused to target proteins for high expression or as an affinity tag for purification, the retention of foreign domains on a target protein may be undesirable, especially for biomedical purposes. Thus, their removal is often crucial at a certain time point after translation. Here, we developed a new strategy to produce target proteins without foreign domains. This involved in vivo removal of foreign domains fused to the N-terminus by the small ubiquitin-related modifier (SUMO) domain/SUMO-specific protease system. This strategy was tested successfully by generating a recombinant gene, BiP:p38:bdSUMO : His:hLIF, that produced human leukemia inhibitory factor (hLIF) fused to p38, a coat protein of the Turnip crinkle virus; the inclusion of p38 increased levels of protein expression. The recombinant protein was expressed at high levels in the leaf tissue of Nicotiana benthamiana. Coexpression of bdSENP1, a SUMO-specific protease, proteolytically released His:hLIF from the full-length recombinant protein in the endoplasmic reticulum of N. benthamiana leaf cells. His:hLIF was purified from leaf extracts via Ni 2+-NTA affinity purification resulting in a yield of 32.49 mg/kg, and the Nterminal 5-residues were verified by amino acid sequencing. Plant-produced His:hLIF was able to maintain the pluripotency of mouse embryonic stem cells. This technique thus provides a novel method of removing foreign domains from a target protein in planta.

Plant produced endotoxin binding recombinant proteins effectively remove endotoxins from protein samples

“…CBM3 tightly binds to microcrystalline cellulose (MCC) beads. The SUMO domain of Brachypodium is used to release the C-terminal region containing CES3 from the full-length recombinant protein by proteolysis, using bdSNEP1, a SUMO protease from Brachypodium 33 . The His-tag of 6 histidine residues (His) was used for western blot analysis and purification using the Ni 2+ -NTA affinity column.…”

“…The upper band corresponds to the full-length recombinant protein. A previous study showed that a recombinant protein containing the SUMO domain is subject to proteolytic cleavage at the C-terminal end of the SUMO domain by an unknown endogenous protease 33 . Thus, BNCS:CES3:His might have been subject to proteolysis at the C-terminal end of the SUMO domain to give a truncated polypeptide at the 51 kD position 33 .…”

Production of Gloeophyllum trabeum Endoglucanase Cel12A in Nicotiana benthamiana for Cellulose Degradation

“…We also incorporated the M-domain of the human receptor-type tyrosine-protein phosphatase C, containing four N-glycosylation sites, as it reportedly enhances the expression of ER-localized proteins remarkably in plants (Kang et al, 2018 ). After the M domain, we added the bdSUMO ( Brachypodium distachyon Small Ubiquitin-like Modifier) and a GG (Gly-Gly) motif, providing the binding site for the protease bdSENP1 ( Brachypodium distachyon Sentrin/SUMO-specific protease-1) for cleavage immediately after the GG motif, thereby helping to achieve the removal of the unnecessary BiP signal peptide, M domain, and bdSUMO after the production of GtCel12A in the ER ( Figure 1A ) (Islam et al, 2019 , 2020 ). After incorporating the GtCel12A coding sequence, we added the coding sequence of CBM3 (family 3 cellulose-binding module) that irreversibly binds to MCC beads and has been previously used as an affinity tag (Islam et al, 2019 ).…”

“…The constructs BiP-M-bdSUMO-GtCel12A-CBM3-HDEL or BiP:bdSENP1:HA (Islam et al, 2020 ) were transformed into Agrobacterium tumefaciens (EHA105). A. tumefaciens cells harboring binary vector constructs were introduced into N. benthamiana leaves via syringe infiltration as described previously (Islam et al, 2019 ; Razzak et al, 2020 ).…”

“…Further, the conditions for plant growth are much less affected by microorganisms that are detrimental to mammalian cell cultures (Buyel et al, 2017 ; Moon et al, 2019 ; Muthamilselvan et al, 2019 ; Knodler and Buyel, 2021 ; Schillberg and Finnern, 2021 ). In addition, previous studies indicated that various useful proteins of different origins, such as bacteria, bacteriophage, animals, and red algae, remained functional when produced in tobacco ( Nicotiana benthamiana ) (Garvey et al, 2013 ; Islam et al, 2019 , 2020 ; Kumari et al, 2020 ; Razzak et al, 2020 ). One of the most important considerations in protein production using plants is how target proteins can be expressed in large amounts because the cost-effectiveness of protein production is often highly dependent on the protein amounts yielded from a unit cultivation area.…”