Journal: Foods

Article Title: Boosting Recombinant Bovine Chymosin in Komagataella phaffii via Fusion Protein and Constitutive Promoter Expression

doi: 10.3390/foods15040731

Figure Lengend Snippet: Purification and deglycosylation analysis of recombinant bovine chymosin. ( a ) SDS-PAGE analysis of samples from the purification process. Proteins were separated on a 12% polyacrylamide gel and stained with Coomassie Brilliant Blue. Approximately 10 µg of protein was loaded per lane. Lane 1: Prestained protein molecular weight markers (sizes indicated on the left). Lane 2: The purified recombinant chymosin obtained after acidification activation (pH 2.5, 2 h) and subsequent concentration using a 5 kDa molecular weight cut-off (MWCO) ultrafiltration membrane. Lane 3: The culture supernatant containing the acid-activated recombinant chymosin prior to ultrafiltration. ( b ) SDS-PAGE analysis of the recombinant chymosin deglycosylation. Analysis was performed under the same conditions as in ( a ). Lane 1: Prestained protein molecular weight markers. Lane 2: Endo-β-N-acetylglucosaminidase H (Endo H) enzyme alone. Lane 3: The purified recombinant chymosin after treatment with Endo H for 2 h at 37 °C, showing the complete deglycosylated form. Lane 4: The purified recombinant chymosin without Endo H treatment, showing the glycosylated form.

Article Snippet: Endo-β-N-acetylglucosaminidase H (Endo H) was obtained from New England Biolabs (Ipswich, MA, USA).

Techniques: Purification, Recombinant, SDS Page, Staining, Molecular Weight, Activation Assay, Concentration Assay, Membrane

Journal: bioRxiv

Article Title: Hyper-Truncated Glycans Augment the Activity of Neutrophil Granule Myeloperoxidase

doi: 10.1101/2020.07.24.219956

Figure Lengend Snippet: A ) i) The MPO N -glycans are positioned distal to the catalytic site. Zoom: Oxidation-prone Met251, Met253 and Trp255 (yellow) in the heme-containing (orange) catalytic site. Mapped on monoprotomeric MPO (PDBID, 1D2V), see for key. ii) Hyper-oxidation of Met251, Met253 and Trp255 indicates extensive auto-oxidation. iii) Se/Pl-MPO from Donor a–b neutrophils displayed a higher chlorination and oxidation activity than MPO from other granules ( Supplementary Table S11 ). Adjusted for MPO levels, n = 3, technical replicates. iv) Relative Tyr chlorination level of proteins from granules fractionated with high resolution of neutrophils from Donor c–f ( Supplementary Table S12 ). Adjusted for total protein and MPO levels, n = 4, biological replicates. B ) i) Endo H-treatment of nMPO produced an Asn355- and Asn391-GlcNAc glycophenotype (mimicking the enriched GlcNAcβAsn signatures of Se/Pl-MPO, see ) as validated using LC-MS/MS . Main glycoforms are depicted for each site. ii) The Endo H-treated nMPO exhibited a higher enzyme activity than native nMPO based on technical triplicate measurements of HOCl production (activity assay 1) and the oxidation rate (activity assays 2–3) ( Supplementary Table S5A-C ). C ) Endo H-treated and untreated nMPO displayed indistinguishable secondary structure profiles based on i) CD profiling ( Supplementary Table S13A-B ) and ii) MD-based predictions. D ) Relative to native nMPO (WT), Endo H-treated MPO (P1) showed i) higher thermal stability as determined by CD 208 nm (arrows indicate initial melting temperatures) and ii) greater global polypeptide accessibility based on MD data ( Supplementary Figure S8-S9 ). Key: a–e and i–ii indicate positions of the MPO glycosylation sites and key catalytic residues, respectively. For panel Bii, Ci, and Di: ^Data from Endo H only controls were subtracted from Endo H-treated nMPO data to enable comparison to untreated nMPO. For all panels: Data plotted as mean ± SD, ns, not significant ( p ≥ 0.05), * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001, ***** p < 0.00005.

Article Snippet: Intact nMPO was incubated with or without Streptomyces plicatus endoglycosidase H (Endo H, Promega) under native conditions, 37°C, 16 h. All samples including controls containing only Endo H were used immediately for activity and inhibition profiling and structural characterisation.

Techniques: Activity Assay, Produced, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: Hyper-Truncated Glycans Augment the Activity of Neutrophil Granule Myeloperoxidase

doi: 10.1101/2020.07.24.219956

Figure Lengend Snippet: A ) Schematics of the glycoform-dependent ceruloplasmin-based inhibition of Endo H-treated and untreated nMPO. Common glycans decorating Asn323, Asn355, and Asn391 in proximity to the MPO-ceruloplasmin interface and the HOCl-producing active site (yellow star) are portrayed. B ) Glycopeptide data (selected EICs) demonstrating complete conversion of Asn355-M6 and Asn391-M6 to GlcNAcβAsn upon Endo H-treatment ( Supplementary Figure S7 ). The Endo H-insensitive Asn323-M2F was included as a control. C ) The chlorination (i) and relative oxidation (ii-iii) levels of native nMPO (left graphs) and Endo H-treated nMPO (right) incubated with (grey bars) and without (black) serum ceruloplasmin (Cp) were determined in technical triplicates using activity assay 1–3, respectively ( Supplementary Table S5D-F ). ^Data from Endo H and Cp only controls were subtracted from Endo H-and Cp-treated nMPO data to enable comparison to untreated nMPO. D ) MD data of Asn323-M2F (green), Asn355-M6 (magenta) and Asn391-M6 (red) modelled on a crystal structure of the ceruloplasmin-MPO complex (PDBID, 4EJX) demonstrated that Asn355-M6 and not Asn355-GlcNAc clashes with ceruloplasmin (see zoom). Data plotted as mean ± SD, ns, not significant ( p ≥ 0.05), * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001.

Article Snippet: Intact nMPO was incubated with or without Streptomyces plicatus endoglycosidase H (Endo H, Promega) under native conditions, 37°C, 16 h. All samples including controls containing only Endo H were used immediately for activity and inhibition profiling and structural characterisation.

Techniques: Inhibition, Incubation, Activity Assay