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Herein, O-GalNAc glycosylated RBD (Tn-RBD) was synthesized as antigen via in vitro glycosylation reactions. The inhibition ability against hACE2 binding of antibodies induced with Tn-RBD was 30–40% increased.

Harnessing highly conserved peptides derived from the receptor binding domain (RBD) of spike (S) protein to construct peptide-based inhibitors is one of the most effective strategies to fight against the ever-mutating coronavirus SARS-CoV-2. But how the O-glycosylation affects their inhibition abilities has not been intensively explored. Herein, an intrinsic O-glycosylated peptide P

Background Therapeutic glycoproteins have occupied an extremely important position in the market of biopharmaceuticals. N-Glycosylation of protein drugs facilitates them to maintain optimal conformations and affect their structural stabilities, serum half-lives and biological efficiencies. Thus homogeneous N-glycoproteins with defined N-glycans are essential in their application in clinic therapeutics. However, there still remain several obstacles to acquire homogeneous N-glycans, such as the high production costs induced by the universal utilization of mammalian cell expression systems, the non-humanized N-glycan structures and the N-glycosylation microheterogeneities between batches. Results In this study, we constructed a Pichia pastoris (Komagataella phaffii) expression system producing truncated N-GlcNAc-modified recombinant proteins through introducing an ENGase isoform (Endo-T) which possesses powerful hydrolytic activities towards high-mannose type N-glycans. The results showed that the location of Endo-T in different subcellular fractions, such as Endoplasmic reticulum (ER), Golgi or cell membrane, affected their hydrolytic efficiencies. When the Endo-T was expressed in Golgi, the secreted IgG1-Fc region was efficiently produced with almost completely truncated N-glycans and the N-GlcNAc modification on the glycosite Asn297 was confirmed via Mass Spectrometry. Conclusion This strategy develops a simple glycoengineered yeast expression system to produce N-GlcNAc modified proteins, which could be further extended to different N-glycan structures. This system would provide a prospective platform for mass production of increasing novel glycoprotein drugs.

Polygonatum sibiricum is a widely used traditional Chinese medicine with various functions. In this study, Bacillus sp. B3 as its endophytic bacterium, which produced the maximum amount of exopolysaccharide (EPS), was isolated. The EPS-B3 produced by Bacillus sp. B3 was purified and identified. As suggested by the analysis of monosaccharide composition, EPS-B3 with an average molecular weight of 1.863×104 Da, was composed of three main monosaccharides, including galactose, glucose and mannose with a molecular ratio of 15: 3: 1. The monosaccharide component of EPS-B3 was different from P. sibiricum polysaccharide (PSP), but they exhibited similar antioxidant and antitumor activity. It is worth to point out that, EPS-B3 exhibited higher Ferric reducing power than PSP. As suggested by the results, the EPS produced by endophytic bacterium from the medicinal plants might have the potential to serve as an alternative of the medicinal herbs.

Additional file 1: Figure S1. SDS-PAGE was used to detect the deglycosylation activity of P. pastoris Pir-Endo-T strain. RNase B (a, upper) and GalNAc-T1 purified from P. pastoris GS115 (b, lower) were used as substrates to incubate at 37 °C for different time. Lane 1: 0 min; Lane 2: 1 h; Lane 3: 2 h; Lane 4: 4 h; Lane 5: 6 h; Lane 6:8 h; Lane 7: over-night; Lane 8: treated with PNGase F 1 h. The star showed the bands from P. pastoris strain. Figure S2. SDS-PAGE analysis of GalNAc-T1 expression in engineered strains at 20 °C. The P. pastoris MNS1-EndoT strain (a and b) and P. pastoris MNN9-EndoT (c and d) were cultured in BMMY with different pH at 20 °C, and 0.5% (a and c) or 1% (b and d) methanol (v/v) was added to the culture every 24 h. Lane 1: 2d; Lane 2: 3d; Lane 3: 4d; Lane 4: 5d. Figure S3. SDS-PAGE analysis of GalNAc-T1 expression in engineered strain at 25 °C. The P. pastoris MNS1-EndoT strain (a and b) and P. pastoris MNN9-EndoT (c and d) was cultured in BMMY with different pH at 25 °C, and 0.5% (a and c) or 1% (b and d) methanol (v/v) was added to the culture every 24 h. Lane 1: 2d; Lane 2: 3d; Lane 3: 4d; Lane 4: 5d. Figure S4. SDS-PAGE analysis of GalNAc-T1 expression in engineered strain. The P. pastoris MNS1-EndoT strain (Right) and P. pastoris MNN9-EndoT (Left) was cultured in BMMY with pH 6.0 at different temperature and different concentration of methanol (v/v) was added to the culture every 24 h. Lane 1: 20 °C 0.5% Methanol 2d; Lane 2: 20 °C 0.5% Methanol, 3d; Lane 3: 25 °C 0.5% Methanol, 2d; Lane 4: 25 °C 0.5% Methanol 3d; Lane 5: 30 °C 0.5% Methanol, 2d; Lane 6: 30 °C 0.5% Methanol, 3d; Lane 7: 20 °C 0.2% Methanol 2d; Lane 8: 20 °C 0.2% Methanol 3d; Lane 9: 20 °C 0.1% Methanol 2d; Lane 10: 20 °C 0.1% Methanol 3d. Figure S5. The purification of IgG1-Fc. IgG1-Fc from E. coli was purified with Ni-NTA and IgG1-Fc from P. pastoris was purified with Protein G column. The numbers showed the different eluted fractions. Figure S6. SDS-PAGE and lectin blot analysis of IgG Fc protein. IgG1-Fc purified from P. pastoris WT (Lane 1) and P. pastoris MNN9-EndoT (Lane 2) were analyzed with Coomassie blue (left) or Con A lectin blot (right). Figure S7. LC/MS-IT-TOF MS analysis of peptide maps from digested recombinant IgG1-Fc proteins. The IgG1-Fc protein from E. coli (upper) and P. pastoris MNN9-EndoT (lower) were digested with Glu-C, and analyzed by LC/MS-IT-TOF. The peak with m/z 713.6287 was assigned as the peptide (P295-QYNSTYRVVSVLTVLHQDWLNGKE-318), while the peak with m/z at 764.3955 was assigned as the peptide (P295–318) with a HexNAc moiety. (4) stands for [M+4H]4+.

The unique type of GTPases in plants, termed ROPs, are the small GTP-binding proteins involved in signal transduction which play important roles in regulation of hormonal response pathway, cell polarity, defense from plant pathogens, etc. In order to explore the regulation mechanism of AtROPs involved in, the purified ROPs were needed to explore the interactions of ROP GTPases with their regulators and effectors. In this study, the first ROP GTPase from Arabidopsis thaliana, AtROP66–178 was successfully expressed in Escherichia coli and obtained in high quality and purity through affinity chromatography and gel-filtration chromatography. The resultant protein was identified as a single band of 19 kDa in SDS-PAGE and was confirmed to be active to interact with guanine nucleotides through the fluorescence-based assay. The intrinsic tryptophan fluorescence intensity of AtROP66–178 was enhanced upon interacting with either GDP or GTP. Meanwhile, the equilibrium dissociation constants of AtROP66–178 with fluorescent guanine nucleotide analogue mantGDP and mantGTP were determined to be 0.0721 μM and 0.0422 μM, respectively, based on fluorescence polarization.

Actin depolymerizing factor (ADF) is a key modulator for dynamic organization of actin cytoskeleton. Interestingly, it was found that the ADF1 gene silencing delays flowering, but its mechanism remains unclear. In this study, ADF1 was used as a bait to screen its interacting proteins by the yeast two-hybrid (Y2H) system. One of them, the REM16 transcription factor was identified. As one of the AP2/B3-like transcriptional factor family members, the REM16 contains two B3 domains and its transcript levels kept increasing during the floral transition stage. Overexpression of REM16 accelerates flowering while silencing of REM16 delays flowering. Gene expression analysis indicated that the key flowering activation genes such as CONSTANS (CO), FLOWERING LOCUS T (FT), LEAFY (LFY) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS (SOC1) were upregulated in the REM16 overexpression lines, while the transcription of the flowering suppression gene FLOWERING LOCUS C (FLC) was decreased. In contrast, the REM16 gene silencing lines contained lower transcript levels of the CO, FT, LFY and SOC1 but higher transcript levels of the FLC compared with the wild-type plants. It was proved that REM16 could directly bind to the promoter regions of SOC1 and FT by in vitro and in vivo assays. Genetic analysis supported that REM16 acts upstream of SOC1 and FT in flowering pathways. All these studies provided strong evidence demonstrating that REM16 promotes flowering by directly activating SOC1 and FT.

Galactokinases, which catalyze the phosphorylation of galactose and possible other monosaccharides, can provide an activated sugar donor to synthesize sugar-containing molecules. In this study, a novel galactokinase from human gut symbiont Akkermansia muciniphila ATCC BAA-835 (GalKAmu) was expressed and characterized. GalKAmu displayed broad substrate tolerance, with catalytic activity towards Gal (100 %), GalN (100 %), GalA (20.2 %), Glc (52.5 %), GlcNAc (15.5 %), Xyl (

The unique type of GTPases in plants, termed ROPs, are the small GTP-binding proteins involved in signal transduction which play important roles in regulation of hormonal response pathway, cell polarity, defense from plant pathogens, etc. In order to explore the regulation mechanism of AtROPs involved in, the purified ROPs were needed to explore the interactions of ROP GTPases with their regulators and effectors. In this study, the first ROP GTPase from Arabidopsis thaliana, AtROP6