Hydroxyproline-O-glycosylated peptide tags enhance recombinant protein yields in tobacco transient expression

Plant transient expression provides a rapid production platform for recombinant proteins but is linked with low protein yields. To test if plant-specific hydroxyproline (Hyp)-O-glycosylated peptide tags attached to a target protein can improve overall yields of recombinant protein transiently expressed in Nicotiana benthamiana, enhanced green fluorescence protein (EGFP) was expressed as a fusion with 5 or 32 tandem repeats of a serine-proline motif, designated (SP)5 or (SP)32, which is known to direct extensive Hyp-O-glycosylation in plants. EGFP containing the (SP)n motif showed enhanced yields in the order as follows: EGFP < EGFP-(SP)5 ≪ (SP)5-EGFP < (SP)32-EGFP. The EGFP equivalent yield of (SP)32-EGFP was up to 16-fold greater than that of the EGFP control. In addition, both fully glycosylated (SP)32-EGFP (∼115 kDa) and partially glycosylated (SP)32-EGFP (∼40 kDa) were detected in protein extracts of N. benthamiana. These two types of glycoforms were completely segregated between media and cells in tobacco BY-2 cell cultures.