Figure 2

Identification of Msp1 as a glycoprotein. (A) SDS-PAGE analysis of LGG's secreted proteins present in spent culture supernatant after overnight growth in AOAC medium. The secreted proteins of LGG WT, msp1 mutant, EPS mutant CMPG5351 and CW-PS mutant CMPG5413 are included. Arrows indicate the major secreted proteins of LGG Msp1 and Msp2 confirmed by Edman degradation and MS/MS. The gels are stained with Sypro^® Ruby and ProQ Emerald glycoprotein stain. (B) Comparative analysis of the glycosylation of Msp1 in spent culture supernatant of LGG wild-type before and after treatment with TFMS, and after recombinant expression in E. coli. The msp1 mutant CMPG10200 was included as a control. This mutant expresses a C-terminally truncated form of Msp1 and lacks the enzymatic active NlpC/P60 domain [17]. SDS-PAGE gels were compared by ConA lectin blotting and blotting with Msp1 specific antiserum. (C) Comparison of the sensitivity of recombinant non-glycosylated Msp1 purified from E. coli (rec. Msp1) and native glycosylated Msp1 purified from LGG WT (glyc. Msp1) towards pronase E and proteinase K. Protein samples were incubated for 1 h at 37°C with the indicated diluted preparations of proteases (Sigma). SDS-PAGE gels were stained with Sypro^® Ruby (Invitrogen). Images were scanned with Typhoon 9400 (GE Healthcare).