FIGURE 2.
Protein glycosylation with the H. influenzae-derived glycan in living E. coli. Glycosylated AcrA acceptor proteins were analyzed by SDS-PAGE and Western blotting. A, periplasmic extracts containing AcrA proteins were analyzed by Western blotting using an anti-AcrA antibody. AcrA from cells containing only the acrA gene (lane 1) or an inactive pglB variant (lane 3) was observed as a single nonglycosylated moiety (−), whereas in the presence of all required functional genes, two additional protein species were produced (lane 2). The monoglycosylated AcrA band is marked with one asterisk, the diglycosylated band with two asterisks. B, glycosylated AcrA containing the H. influenzae-derived glycan was purified by nickel affinity chromatography and analyzed by SDS-PAGE and Coomassie staining.