Journal: Microbial Cell Factories

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

doi: 10.1186/s12934-021-01689-x

Figure Lengend Snippet: Structural variation of model glycoproteins. A Structural models of (i) NGRP, (ii) scFv13R4 and scFv13R4CM were generated by Phyre2 based on protein homology prediction (≥ 99% confidence) . (iii) X-ray crystallographic structure of RNase A (PDB code 3WMR). Ribbon model of the proteins was drawn by UCSF Chimera . Position of the sequon variants (D/E-X-N-X-S/T, X ≠ P) within the protein is indicated. Disulphide-bonds are highlighted in yellow. C-terminal sequon (DQNAT) of scFv13R4 is not displayed in the protein model. B Linear representation of proteins; (i) NGRP, (ii) scFv13R4, (iii) scFv13R4CM, and (iv) RNase A. Position of disulphide-bonds (C–C) and glycosylation sites (N) are indicated with amino acid positions

Article Snippet: RnaseAlert lab test kit (Invitrogen) was used to assay RNase A activity of the protein sample.

Techniques: Generated

Journal: Microbial Cell Factories

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

doi: 10.1186/s12934-021-01689-x

Figure Lengend Snippet: Effect of culture conditions and oxygen availability upon target protein glycosylation in E. coli . A – D Effect of culture to flask volume ratio (oxygen transfer efficiency) upon target protein glycosylation in E. coli . Quantitative Western blot analysis (densitometry) of A scFv13R4, B scFv13R4CM, C RNase A and D non-disulphide control protein NGRP located in periplasm of glyco-competent E. coli in shake flask under three different cultures to flask volume ratio (5:50, 10:50, and 25:50 ml). E – H Quantitative Western blot analysis (densitometry) of E scFv13R4, F scFv13R4CM, G RNase A and H NGRP control non-disulphide bond-containing protein, detected in the periplasm of glyco-competent E. coli under different oxygen levels culture (3% and 15% O 2 ). A – H Total proteins 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 OD 600 of the samples. A – H Glycosylated (yellow bar) and non-glycosylated (green bar) protein as shown (left y-axis). % Glycosylation (% G 1 /G 0 + G 1 ) is indicated (black circle, right y-axis). Statistical analysis was conducted by unpaired t-test with Welch’s correction to control sample at lowest culture to flask volume ratio 5:50 ( A – D ) or to control normoxic culture ( E–H ) ( P < 0.05*, < 0.01**, for % glycosylation; P < 0.05 ◊ , for total protein mg/g DCW). All data were processed from three biological replicates. Error bars indicate standard deviation from mean values

Article Snippet: RnaseAlert lab test kit (Invitrogen) was used to assay RNase A activity of the protein sample.

Techniques: Western Blot, Purification, Standard Deviation

Journal: Microbial Cell Factories

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

doi: 10.1186/s12934-021-01689-x

Figure Lengend Snippet: Comparative cell growth and volumetric productivity (protein titre) during recombinant disulphide-bond protein production in oxygen-depleted conditions

Article Snippet: RnaseAlert lab test kit (Invitrogen) was used to assay RNase A activity of the protein sample.

Techniques: Recombinant

Journal: Microbial Cell Factories

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

doi: 10.1186/s12934-021-01689-x

Figure Lengend Snippet: Glycosylation of disulphide bond-containing proteins in oxidoreductase mutant (Δ dsbB ) of E. coli . Quantitative Western blot analysis (densitometry) of A scFv13R4, B scFv13R4CM, C RNase A and D control non-disulphide bond-containing protein NGRP expressed in periplasmic of glyco-competent E. coli wild-type ( wt ) or Δ dsbB strain. Total proteins ( A – D ) were quantified using pre-determined purified scFv13R4CM, RNase A, or NGRP standard curve (5 ng to 100 ng). The data were converted into yield mg/g of dry cell weight (DCW) based on normalisation and calculation with measured OD 600 of the samples Glycosylated (yellow bar) and non-glycosylated (green bar) protein as shown (left y-axis). % Glycosylation (% G /G 0 + G ) is indicated (black circle, right y-axis). Additional file : Figure S16, A and B showed representative Western blot images for scFv13R4CM and RNase A produced in wt and Δ dsbB strains. Representative Western blot image for NGRP and scFv13R4 have been shown before in Figs. B, C, and B, C. Statistical analysis was conducted by unpaired t-test with Welch’s correction to control sample expressed in wt strain ( P < 0.05*, < 0.01**, for % glycosylation; P < 0.05 ◊ , < 0.01 ◊◊ , < 0.0001 ◊◊◊◊ , for total protein mg/g DCW). All data were processed from three biological replicates. Error bars indicate standard deviation from mean values

Article Snippet: RnaseAlert lab test kit (Invitrogen) was used to assay RNase A activity of the protein sample.

Techniques: Mutagenesis, Western Blot, Purification, Produced, Standard Deviation

Journal: Microbial Cell Factories

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

doi: 10.1186/s12934-021-01689-x

Figure Lengend Snippet: Comparative cell growth and volumetric production (protein titre) during recombinant disulphide-bond protein production in the wild-type ( wt ) and Δ dsbB strain

Article Snippet: RnaseAlert lab test kit (Invitrogen) was used to assay RNase A activity of the protein sample.

Techniques: Recombinant

Journal: Microbial Cell Factories

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

doi: 10.1186/s12934-021-01689-x

Figure Lengend Snippet: Impact of cystine supplementation upon glycosylation of recombinant proteins in Δ dsbB strain and glycosylation of RNase A in disulphide-bond isomerase mutant (Δ dsbC ). (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 Δ dsbB 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 Δ dsbC . Total proteins ( A – F ) 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 OD 600 of the samples. Glycosylated (yellow bar) and non-glycosylated (green bar) protein as shown (left y-axis). % Glycosylation (% G 1 /G 0 + G 1 ) 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 Δ dsbB strain without cystine treatment ( A – D ) 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

Article Snippet: RnaseAlert lab test kit (Invitrogen) was used to assay RNase A activity of the protein sample.

Techniques: Recombinant, Mutagenesis, Western Blot, Produced, Expressing, Purification, Standard Deviation

Journal: Microbial Cell Factories

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

doi: 10.1186/s12934-021-01689-x

Figure Lengend Snippet: Comparative cell growth and volumetric production (protein titre) during recombinant disulphide-bond protein production in the wild-type ( wt ) and Δ dsbB strain supplemented with small molecule oxidant (cystine)

Article Snippet: RnaseAlert lab test kit (Invitrogen) was used to assay RNase A activity of the protein sample.

Techniques: Recombinant

Journal: Microbial Cell Factories

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

doi: 10.1186/s12934-021-01689-x

Figure Lengend Snippet: Comparative cell growth and volumetric production (protein titre) during RNase A production in the wild-type ( wt ) and Δ dsbC strain

Article Snippet: RnaseAlert lab test kit (Invitrogen) was used to assay RNase A activity of the protein sample.

Techniques:

Journal: Microbial Cell Factories

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

doi: 10.1186/s12934-021-01689-x

Figure Lengend Snippet: Summary of glycosylation efficiency (%) and glycoprotein titre (mg/L of glycosylated protein) of A scFv13R4, B scFv13R4CM, C RNase A, and D NGRP during production in glyco-competent E. coli K-12 by use of different cultivation conditions to modulate target protein folding. Glycoprotein titres were converted from the yield (mg/g DCW), cell growth, and total protein titre results given in the Figs. , , , and Tables , , , and Additional file : Table S6 (“ ” section). Colour symbols indicate the experiment or figure/table sources for the data (Fig; = Table , = Table , = Table , = Table , = Additional file : Table S6). A similar wt and Δ dsbB cultivation were run in four different batches of experiment, which were (i) wt oxygen transfer experiment (10/50, Fig. A–D), (ii) wt against oxidoreductase knockout Δ dsbB (Fig. A–D), (iii) wt and ΔdsbB 0 cystine treatment (Fig. A–D), (iv) wt against oxidoreductase knockout Δ dsbC (Fig. E, , Additional file : Figure S18A, B). A variation of inducer concentration for NGRP expression was used in the experiment of Fig. D (200 μM instead of 40 μM PPDA, “ ” section)

Article Snippet: RnaseAlert lab test kit (Invitrogen) was used to assay RNase A activity of the protein sample.

Techniques: Knock-Out, Concentration Assay, Expressing