C-Terminally fused affinity Strep-tag II is removed by proteolysis from recombinant human erythropoietin expressed in transgenic tobacco plants

Abstract

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-rhuEPOP). To facilitate purification from plant extracts, Strep-tag II was engineered at the C-terminus of EPO. Although asialo-rhuEPOP 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-rhuEPOP 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-rhuEPOP 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-rhuEPOP. However, Strep-tag II was detected on asialo-rhuEPOP 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.

Progress toward Eukaryotic Semisynthetic Organisms: Translation of Unnatural Codons

“…From control cells transfected with natural EGFP mRNA, a dominant band was observed with a faster migrating, weaker band (Figure 3). The faster migrating band was attributed to partial Strep tag degradation 21 (Figure S1). As expected, neither band showed any TAMRA signal.…”