Abstract
SummaryPlants 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 (CBM3), 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 (hIL6) with a yield of 18.49 mg/kg fresh weight from leaf extracts of Nicotiana benthamiana following Agrobacterium‐mediated transient expression. Plant‐produced hIL6 (P‐hIL6) contained less than 0.2 EU/μg (0.02 ng/mL) endotoxin. P‐hIL6 activated the Janus kinase‐signal transducer and activator of transcriptional pathways in human LNCaP cells, and induced expression of IL‐21 in activated mouse CD4+ T cells. This approach is thus a powerful method for producing recombinant proteins in plants.
Highlights
The commercialization of recombinant proteins is a mainstream area in biotechnology
CBM3-tagged MerP (CBM3) binds to the surface of microcrystalline cellulose (MCC) (Hong et al, 2007) and inexpensive cellulose beads can be used as an affinity resin
As we used Agrobacterium-mediated transient expression to express human interleukin-6 (hIL6) in plants, we examined the endotoxin content in hIL6 purified from plant tissue using the chromogenic kinetic method based on the Limulus amebocyte lysate assay (LAL-test). hIL6 purified from plants contained
Summary
The commercialization of recombinant proteins is a mainstream area in biotechnology. The most powerful is transient expression following Agrobacterium-mediated gene transfer into host plants via RNA virus-mediated RNA amplification or DNA virus-based gene amplification. Another important approach is chloroplast transformation, which integrates the target gene into the chloroplast genome and thereby leads to extremely high levels of protein production (Staub et al, 2000; Verma and Daniell, 2007; Zhang et al, 2017); the transgenic approach, which integrates foreign genes into the plant nuclear genome, is widely used (Lee et al, 2015; Sohn et al, 2018; Twyman et al, 2002). If whole plants are used, different tissues can be employed to produce recombinant proteins
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