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 (similar to 115 kDa) and partially glycosylated (SP)(32)-EGFP (similar to 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. (C) 2013 Elsevier Ltd. All rights reserved.