Journal: ACS Omega

Article Title: Direct MALDI Glycotyping of Glycoproteins toward Practical Subtyping of Biological Samples

doi: 10.1021/acsomega.2c05429

Figure Lengend Snippet: (a) 0,2 A, 2,4 A, and B type ISD fragment position of N -glycans. MALDI–ISD spectrum of RNase B (20 pmol μL –1 ) with (b) 1,5-diaminonaphthalene (DAN)/DHB/Na (12:10:1), (c) DAN/aniline/DHB/Na (2:10:10:1), and (d) DAN/DHB/Na (2:10:1).

Article Snippet: Ribonuclease B (RNase B) from bovine was purchased from New England BioLabs, Inc. (Beverly, MA, USA).

Techniques:

Journal: PLoS ONE

Article Title: The pCri System: A Vector Collection for Recombinant Protein Expression and Purification

doi: 10.1371/journal.pone.0112643

Figure Lengend Snippet: ( A ) The GFP gene was cloned into pCri-1a, 4a, 6a, 8a, 11a, and 14a, the proteins expressed in E. coli BL21 cells, and subsequently purified by Ni-NTA-affinity chromatography except for MBP, GST, and LSL fusion products, which were purified by their respective specific affinity resins. ( B ) The gene coding for fragilysin was cloned into pCri-1a, 4a, 6a and 8a, and expressed in E. coli Origami 2 cells. Total (T) and soluble (S) fractions of crude protein extracts were further analysed by SDS-PAGE. All expression trials were performed at 20°C except for pCri-1a, which was also performed at 37°C. ( C ) Partially purified MBP-fragilysin before (−) and after (+) TEV proteinase cleavage. Arrows indicate the soluble fraction of fragilysin (white) and the MBP (black) after TEV proteinase cleavage. ( D ) Expression of CPA2 intracellularly (lanes 1 and 2) or periplasmatically (lanes 3 and 4) in E. coli cells, and extracellularly (lanes 5 and 6) in P. pastoris cells. Lanes indicate samples before (1, 3 and 5) and after (2, 4 and 6) tryptic digestion. Arrows indicate the pro-CPA2 (black), the mature form (grey) and the pro-peptide (white) after tryptic cleavage. ( E ) The PNGase F gene was cloned into pCri-4a and 8a and expressed overnight at 20°C in E. coli BL21 and Origami 2 cells. Total (T) and soluble (S) fractions of crude protein extracts were further analysed by SDS-PAGE. ( F ) Activity of affinity-purified TRX-PNGase F against glycosylated RNase B. (+) and (−) indicate presence and absence of PNGase F. Arrows indicate the PNGase F (black), native RNase B (grey) and deglycosylated RNase B (white). ( G ) MecR1 was expressed in E. coli BL21 using pCri-8a or 13a, and soluble fractions were analysed by Western blotting with specific antibodies as detailed in “ ”. A black arrow indicates the detected MecR1. ( H ) Partially purified MISTIC-MecR1 after Ni-NTA-affinity purification. ( I ) Partially purified MBP-GFP, SUMO-GFP and MISTIC-MecR1 were digested with TEV proteinase, SENP1 or thrombin, respectively. For TEV proteinase and SENP1 digestions various ratios of proteinase∶tagged-protein were tested in overnight incubations at 4°C, whereas for thrombin digestions 2 units of proteinase were used to digest 25 µg of protein for various times at room temperature. Arrows indicate tagged-protein (black), target protein (grey) and fused-tag (white) after proteinase cleavage.

Article Snippet: Glycosidase activity of PNGase F was tested against the glycoprotein ribonuclease B (RNase B; New England Biolabs) at a w/w ratio of 1∶5 PNGase F/RNase B and a final protein concentration of 0.5 mg/mL.

Techniques: Clone Assay, Purification, Affinity Chromatography, SDS Page, Expressing, Activity Assay, Affinity Purification, Western Blot

Journal: bioRxiv

Article Title: A novel PNGase Rc for improved protein N-deglycosylation in bioanalytics and HDX-MS epitope mapping under challenging conditions

doi: 10.1101/2022.04.13.488165

Figure Lengend Snippet: ( a ) HRP (I), RNase B (II) or fetuin (III) were incubated for various times with PNGase Rc or F at pH 3.5 or 7.5, respectively. Deglycosylation was performed at an E:S of 1:48 (M:M) and at 37 °C. Deglycosylation efficiencies were monitored by SDS-PAGE followed by coomassie staining at indicated time points. ( b ) m/z spectra of intact SIRPα after overnight incubation at 37 °C and an E:S of 1:45 with PNGase Rc (I) or PNGase F (II), at pH 2.5 or pH 7.4 respectively. The inlayer of the lower panel shows the charge-deconvoluted mass spectrum confirming the identity of SIRPα.

Article Snippet: Trastuzumab was kindly donated by MAB Discovery GmbH (Polling, Germany), fetuin from fetal calf serum (New England Biolabs, Ipswich MA, USA), RNase B from bovine pancreas (New England Biolabs, Ipswich MA, USA), and horse radish peroxidase (HRP, Sigma-Aldrich, Munich, Germany), PNGase F from F. meningosepticum (R&D Systems, Wiesbaden, Germany), human SIRPα (Glu 31 - Arg 370 (ACROBiosystems, Newark, DE, USA)

Techniques: Incubation, SDS Page, Staining

Journal: Frontiers in Microbiology

Article Title: Expression of Human ACE2 N-terminal Domain, Part of the Receptor for SARS-CoV-2, in Fusion With Maltose-Binding Protein, E. coli Ribonuclease I and Human RNase A

doi: 10.3389/fmicb.2021.660149

Figure Lengend Snippet: Escherichia coli expression strains, purification method, and enzyme activity/potential usage.

Article Snippet: MBP-RNase I b , NEB Turbo or NEB Express , Amylose column , Cleaving 300-nt RNA.

Techniques: Expressing, Purification, Activity Assay, Magnetic Beads, Binding Assay, Fluorescence

Journal: bioRxiv

Article Title: Systems-wide analysis of glycoprotein conformational changes by limited deglycosylation assay

doi: 10.1101/2021.06.04.447131

Figure Lengend Snippet: Experimental conditions for optimal limited deglycosylation of bovine RNase B and fetuin by PNGase F were determined. The optimal A ) NP-40 concentration, B ) temperature, C ) and D ) PNGase F:glycoprotein ratio for RNase B and fetuin, respectively, and, E ) and F ) the optimal time for the deglycosylation of RNase B and fetuin, respectively, were assayed by differential migration of the glycoproteins in 15% SDS-PAGE. Fully deglycosylated (+) and fully glycosylated (−) proteins were included for each optimization reaction, denoted by FD and FG, respectively.

Article Snippet: Proof-of-concept and optimization of the LDA method were performed using two standard glycoproteins, bovine RNase B and fetuin (New England Biolabs) by PNGase F (New England Biolabs) under native conditions.

Techniques: Concentration Assay, Migration, SDS Page