Figure 6.

Characterization of LTBMUC1 produced in transgenic N. benthamiana L. plants expressing either human GalNAc-T2 or co-expressing human GalNAc-T2, Y. enterocolitica UDP-GlcNAc-4 epimerase, and C. elegans UDPGlcNAc/UDPGalNAc transporter. A - amino acid sequence of His-tagged LTBMUC1 fusion protein: MUC1 sequence is underlined, GPGP linker is italisized, experimentally confirmed sites for attachment of GalNAc by GalNAc-T2 are red-highlighted, covered regions after LC-MS/MS analysis of the chymotryptic fragments are gray-highlighted; B - purification of LTBMUC1 produced in GalNAc-T2 and GalNAc-T2//UDP-GlcNAc-4 epimerase//UDP-GlcNAc/UPD-GalNAc transporter plants by Ni²⁺ Sepharose Fast Flow chromatography: Fractions (1, 2, 3, 4 for LTBMUC1 produced in GalNAc-T2 plants; and 1', 2', 3', 4' for LTBMUC1 produced in the triple transgenic plants) eluted from the resin with 20 mM phosphate buffer pH 7.4 containing 0.5 M NaCl and 0.5 M imidazol, were screened by HisProbe-HRP (upper panels); the same blots were stained in parallel with Ponceau S (lower panels); C - VVA chromatography of LTBMUC1 produced in the tested plant systems after purification on Ni²⁺ Sepharose Fast Flow: identical aliquots of the volume-equalized samples 2 and 2' were screened by HisProbe-HRP before loading on VVA agarose gel (panel HisProbe-HRP detection before VVA agarose); bound proteins were eluted from the resin with 50 mM Tris HCl buffer pH 7.4 containing 0.1 M NaCl, 1 mM MgCl₂ and 0.3 M GalNAc, and identical aliquots were screened by HisProbe-HRP (panel HisProbe-HRP detection after VVA agarose), VVA blotting (panel VVA blot after VVA agarose), and SDS-PAGE with subsequent silver-staining (panel silver staining after VVA agarose); asterisk denotes the position of LTBMUC1; D - schematic diagram of T-DNA of pBYR:GNE.GT plant binary expression vector. Abbreviations: LB, left border; RB, right border; Pnos, nopaline synthase promoter; P35 S, CaMV 35 S promoter; nptII, neomycin phosphotransferase gene; gne - Y. enterocolitica UDP-GlcNAc-4-epimerase gene; UDP-GalNAc - C. elegans UDP-GlcNAc/UDP-GalNAc transporter gene; Tnos, 3' termination signal of nopaline synthase gene; T35 S, 3' termination signal of CaMV 35 S gene; TvspB - 3' termination signal of soybean vegetative storage protein B gene; TrbcS - 3' termination signal of Rubisco small subunit gene; TEV HT - etch virus translational enhancer element; E - RT-PCR confirmation of the co-expression of human GalNAc-T2 (GalNAc-T2), C. elegans UDP-GlcNAc/UPD-GalNAc transporter (transporter), and Y. enetrocolitica UDP-GlcNAc-4 epimerase (gne) in N. benthamana L. plants using 0.5 μg total RNA as a template and primers G3/G4, GT3/GT4, and GE4/GE5, respectively (see "Materials and Methods). F - spectrum of a chymotryptic peptide of the 20/21 kDa band denoted in C by an asterisk, assisting in the identification of the band as LTBMUC1.