Fig. 3
Glycosylation of NGRP in glyco-competent E. coli. A Organisation of pDEST-ORS expression vector used in this study. Target gene (NGRP) was fused with Sec signal peptide PelB (N-terminal) and Hexahistidine-tag (C-terminal). Expression of the target gene was regulated transcriptionally by Ptac via IPTG induction and translationally by orthogonal riboswitch (ORS) via PPDA titration. IPTG = Isopropyl β-d-1-thiogalactopyranoside, PPDA = Pyrimido [4,5-d] pyrimidine-2,4-diamine. Three synonymous nucleotide sequence variants of PelB-NGRP N-terminal codon (PelB 1, PelB 2, and PelB WT or wild-type) are shown. These variants were tested to explore the impact of the 5ʹ codon context upon the riboswitch-dependent regulatory function. B Western blot analysis of periplasmic fractions of glyco-competent (GC) and non-glycocompetent (Non-GC) strains of E. coli. Anti-His antibody was used to detect the NGRP. Arrows indicate non-glycosylated (G0) and glycosylated (G1) NGRP. C, D Quantitative Western blot analysis (Densitometry) of NGRP located in the periplasmic fractions from the two signal peptide variants, C PelB 1 and D PelB 2-NGRP. Proteins were produced with increasing PPDA inducer concentrations (100 μM IPTG and 0, 2, 8, 40, 100, 200, 400 μM PPDA). Total proteins were quantified using a pre-determined purified NGRP standard curve (15 ng to 100 ng) (“Methods”). The data were converted into mg/g of dry cell weight (DCW) based on normalisation and calculation with measured OD600 of the samples. Glycosylated (yellow bar) and non-glycosylated (green bar) protein are as shown (left y-axis). % Glycosylation (% G1/G0 + G1) is indicated (black circle, right y-axis). Data were processed from three biological replicates; error bars represent standard deviation from mean values. Representative Western blots are shown as insets