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Fig. 7 | Microbial Cell Factories

Fig. 7

From: Genetic and process engineering strategies for enhanced recombinant N-glycoprotein production in bacteria

Fig. 7

Impact of cystine supplementation upon glycosylation of recombinant proteins in ΔdsbB strain and glycosylation of RNase A in disulphide-bond isomerase mutant (ΔdsbC). (A-D) Quantitative Western blot analysis (densitometry) of A scFv13R4, B scFv13R4CM, C RNase A and D NGRP non-disulphide control protein produced in the periplasm of glyco-competent E. coli wild-type (wt) or ΔdsbB strain supplemented with or without 100 μM cystine during protein expression. E, F Quantitative Western blot analysis (densitometry) of E RNase A and F control non-disulphide bond-containing protein NGRP expressed in periplasmic of glyco-competent E. coli ΔdsbC. Total proteins (AF) were quantified using pre-determined purified scFv13R4CM, RNase, or NGRP standard curve (5 ng to 100 ng). 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). Statistical analysis was conducted by unpaired t-test with Welch’s correction to control sample expressed in wt or ΔdsbB strain without cystine treatment (AD) or to control sample expressed in wt strain (E, F) (P < 0.05*, < 0.01**, for % glycosylation; P < 0.05, < 0.001, for normalised total protein). All data were processed from three biological replicates. Error bars indicate standard deviation from mean values

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