title: C-Terminally fused affinity Strep-tag II is removed by proteolysis from recombinant human erythropoietin expressed in transgenic tobacco plants.
abstract: C -terminally fused Strep -tag II is removed from rhuEPO expressed in tobacco plants. The finding suggests that direct fusion of purification tags at the C -terminus of rhuEPO should be avoided. Asialo-erythropoietin (asialo-EPO), a desialylated form of EPO, is a potent tissue-protective agent. Recently, we and others have exploited a low-cost plant-based expression system to produce recombinant human asialo-EPO (asialo-rhuEPO(P)). To facilitate purification from plant extracts, Strep-tag II was engineered at the C-terminus of EPO. Although asialo-rhuEPO(P) was efficiently expressed in transgenic tobacco plants, affinity purification based on Strep -tag II did not result in the recovery of the protein. In this study, we investigated the stability of Strep-tag II tagged asialo-rhuEPO(P) expressed in tobacco plants to understand whether this fused tag is cleaved or inaccessible. Sequencing RT-PCR products confirmed that fused DNA sequences encoding Strep-tag II were properly transcribed, and three-dimensional protein structure model revealed that the tag must be fully accessible. However, Western blot analysis of leaf extracts and purified asialo-rhuEPO(P) revealed that the Strep-tag II was absent on the protein. Additionally, no peptide fragment containing Strep-tag II was identified in the LC-MS/MS analysis of purified protein further supporting that the affinity tag was absent on asialo-rhuEPO(P). However, Strep-tag II was detected on asialo-rhuEPO(P) that was retained in the endoplasmic reticulum, suggesting that the Strep-tag II is removed during protein secretion or extraction. These findings together with recent reports that C-terminally fused Strep-tag II or IgG Fc domain are also removed from EPO in tobacco plants, suggest that its C-terminus may be highly susceptible to proteolysis in tobacco plants. Therefore, direct fusion of purification tags at the C-terminus of EPO should be avoided while expressing it in tobacco plants.
authors.affiliation: Department of Pharmaceutical Sciences, Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, 1801 Fayetteville Street, Durham, NC, 27707, USA.
authors.forename: Farooqahmed S
authors.initials: FS
authors.lastname: Kittur
authors.forename: Mallikarjun
authors.initials: M
authors.lastname: Lalgondar
authors.forename: Chiu-Yueh
authors.initials: CY
authors.lastname: Hung
authors.forename: David C
authors.initials: DC
authors.lastname: Sane
authors.forename: Jiahua
authors.initials: J
authors.lastname: Xie
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D000068817:Epoetin Alfa / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism*; D030821:Plants, Genetically Modified; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014026:Nicotiana / Q000235:genetics*
chemical_list: D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; C583458:Trp-Ser- His-Pro-Gln-Phe-Glu-Lys; D000068817:Epoetin Alfa