Fig. 4

Production of NGRP and scFv13R4 isoforms containing signal peptide cleavage site variants. A Schematic representation of four predicted isoforms of NGRP or scFv13R4 (a-d-forms) based on their signal peptide processivity and protein glycosylation. B, C Western blot analysis of membrane (M) and periplasmic (P) expression of wild type (wt) and signal peptide cleavage mutant (TMT) NGRP (B) and scFv13R4 (C) in glyco-competent (GC) and non glyco-competent (Non-GC) E. coli. B, C All NGRP and scFv13R4 isoforms were detected by anti-His antibody. Glycosylated scFv13R4 was detected by CjNgp antibodies. Predicted a-d isoforms within the bands are indicated. The lanes in panel B are from the same blot while white vertical lines indicates a non-adjacent lanes. The protein migration band relative to each other in different lanes in the blot is unchanged. Additional file 1: Figure S7 shows the uncropped Western blot image of B. D–G Quantitative Western blot analysis (Densitometry) of membrane (TMT) and periplasmic (wt) localised D–E NGRP and F–G scFv13R4 produced in glyco-competent E. coli. Proteins were produced under different induction conditions (100 μM IPTG and 0, 8, 40 and 200 μM PPDA). Anti-His antibody was used to detect the proteins (glycosylated and non-glycosylated). Total proteins were quantified using a pre-determined purified NGRP or scFv13R4 standard curve (5 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 as shown (left y-axis). % Glycosylation (% G₁/G₀ + G₁) is indicated (black circle, right y-axis). All data (D–G) were processed from three biological replicates. Error bars indicate standard deviation from mean values