Expression and <i>in vitro</i> glycosylation of recombinant edible bird nest (EBN) mucin

Ge Cheng; Yunsheng Lyu; Ran Ran; Li Liu; Josef Voglmeir; Ge Cheng; Yunsheng Lyu; Ran Ran; Li Liu; Josef Voglmeir

doi:10.48130/fmr-0023-0037

Figure 1.

EBN is produced by male swiftlets' salivary glands. The largest portion of this dried saliva consists of mucins, which are proteins heavily decorated with complex carbohydrates linked via N-acetylgalactosamine to the AfMuc protein backbone.

Figure 2.

Activity of AfGalNT2 and HsGalNT2 towards the Muc1 peptide substrate. Reaction mixtures containing the Muc1 peptide in the presence of UDP-GalNAc and (a) AfGalNT2; (b) HsGalNT2; (c) no enzyme; (d) Galactosylation reaction of the GalNAc-Muc1 glycopeptide substrate in the presence of CjCgTB30 enzyme and UDP-galactose; (e) reaction mixtures without CjCgTB30.

Figure 3.

Biochemical characterization of AfGalNT2 and HsGalNT2. The following parameters were measured for both enzymes using the EA2 peptide: (a) pH optimum, (b) temperature optimum and (c) metal ion dependence.

Figure 4.

Purification and in vitro glycosylation of AfMuc. (a) SDS-PAGE analysis of AfMuc during expression and purification. The red arrow indicates the position of the desired purified protein. (b) MALDI-ToF MS analysis of recombinant AfMuc without the presence of HsGalNT2. (c) MALDI-ToF MS analysis of recombinant AfMuc in the presence of HsGalNT2. The intermediate-mass peaks between discrete GalNAc units may be the result of terminal proteolysis of the recombinant AfMuc protein backbone.