Cost‐effective production of tag‐less recombinant protein inNicotiana benthamiana

Abstract

Summary Plants have recently received a great deal of attention as a means of producing recombinant proteins. Despite this, a limited number of recombinant proteins are currently on the market and, if plants are to be more widely used, a cost‐effective and efficient purification method is urgently needed. Although affinity tags are convenient tools for protein purification, the presence of a tag on the recombinant protein is undesirable for many applications. A cost‐effective method of purification using an affinity tag and the removal of the tag after purification has been developed. The family 3 cellulose‐binding domain ( CBM 3), which binds to microcrystalline cellulose, served as the affinity tag and the small ubiquitin‐related modifier ( SUMO ) and SUMO ‐specific protease were used to remove it. This method, together with size‐exclusion chromatography, enabled purification of human interleukin‐6 ( hIL 6) with a yield of 18.49 mg/kg fresh weight from leaf extracts of Nicotiana benthamiana following Agrobacterium ‐mediated transient expression. Plant‐produced hIL 6 (P‐ hIL 6) contained less than 0.2 EU/μg (0.02 ng/mL) endotoxin. P‐ hIL 6 activated the Janus kinase‐signal transducer and activator of transcriptional pathways in human LNC aP cells, and induced expression of IL ‐21 in activated mouse CD 4 + T cells. This approach is thus a powerful method for producing recombinant proteins in plants.

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

“…The PCR products were digested with BamHI and XhoI restriction endonucleases and introduced into the plant expression vector, 326-GFP which had been digested with the same restriction endonucleases, to give p326-His:bdSENP1 ( Figure 1A) and p326-bdSENP1:HA. The synthetic hLIF gene was incorporated to the C-terminus of M:CBM3:bdSUMO by overlapping PCR using primers csLF-1, csLR-2, csLF-3, and csLR-4 ( Supplementary Table S1) using 326-M:CBM3: bdSUMO:hIL6 as a template (Islam et al, 2019a). Six histidine residues (His) were added at the N-terminus of hLIF (His:hLIF) during PCR amplification.…”

“…In addition to such well-established enzymes, a highly active proteolytic enzyme, SUMO-specific protease, derived from Brachypodium distachyon (B. distachyon) has recently been used to remove a foreign domain in vitro (Frey & Görlich, 2014;Islam et al, 2019a). This protease recognizes the entire SUMO domain (Bailey and O'Hare, 2004;Hickey et al, 2012), and thus there is no possibility of nonspecific cleavage of the protein substrate.…”

“…This protease recognizes the entire SUMO domain (Bailey and O'Hare, 2004;Hickey et al, 2012), and thus there is no possibility of nonspecific cleavage of the protein substrate. Moreover, SUMO-specific protease leaves no extra residues on the target protein after cleavage (Hickey et al, 2012;Islam et al, 2019a).…”

Structural Biology and Electron Microscopy of the Autophagy Molecular Machinery

“…Compared to their yeast and mammalian counterparts, plants contain some additional homologs or plant-specific components, the function of which remain to be explored [18][19][20][21][22][23]. It is noteworthy that biochemical, structural, and functional studies of plant ATG proteins are severely hampered by the difficulty in protein production, a challenge often encountered when working in plant proteins in general [24]. There exists several technologies dedicated to producing proteins in plants, including the Agrobacterium-mediated transient gene expression [24], chloroplast transformation [25], and stable transformation that integrates foreign genes into the plant nuclear genome [26].…”

“…It is noteworthy that biochemical, structural, and functional studies of plant ATG proteins are severely hampered by the difficulty in protein production, a challenge often encountered when working in plant proteins in general [24]. There exists several technologies dedicated to producing proteins in plants, including the Agrobacterium-mediated transient gene expression [24], chloroplast transformation [25], and stable transformation that integrates foreign genes into the plant nuclear genome [26]. Unfortunately, both the transient expression and the chloroplast transformation are of only limited use at present owing to the inability to either express proteins of large size or in sufficient quantity, which is particularly important for structural studies.…”