Glycosylation and antiproliferative activity of hyperglycosylated IFN-α2 potentiate HEK293 cells as biofactories

Gugliotta, Agustina; Ceaglio, Natalia; Raud, Brenda; Forno, Guillermina; Mauro, Laura V.; Kratje, Ricardo; Oggero, Marcos

doi:10.1016/j.ejpb.2016.11.012

Cited by 13 publications

(7 citation statements)

References 53 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Two additional heterogeneous glycosylation patterns were included, based on Watanabe et al 12 (pat1) and Shajahan et al (pat2) 11 . However, it is important to note glycosylation patterns and glycan species can vary in different cell types 15 and are altered in malignant cells compared to nonmalignant cells 16 . As these previous S-protein glycosylation studies were performed in HEK293 cells 11,12 , which were immortalised by adenovirus, it is possible that the glycosylation pattern may not reflect a natural infected cell.…”

Section: N-glycosylation Sites In the Sars-cov-2 S-proteinmentioning

confidence: 99%

Impact of glycan cloud on the B-cell epitope prediction of SARS-CoV-2 Spike protein

Wintjens

Bifani

2020

npj Vaccines

View full text Add to dashboard Cite

The SARS-CoV-2 outbreak originated in China in late 2019 and has since spread to pandemic proportions. Diagnostics, therapeutics and vaccines are urgently needed. We model the trimeric Spike protein, including flexible loops and all N-glycosylation sites, in order to elucidate accessible epitopes for antibody-based diagnostics, therapeutics and vaccine development. Based on published experimental data, six homogeneous glycosylation patterns and two heterogeneous ones were used for the analysis. The glycan chains alter the accessible surface areas on the S-protein, impeding antibody-antigen recognition. In presence of glycan, epitopes on the S1 subunit, that notably contains the receptor binding domain, remain mostly accessible to antibodies while those present on the S2 subunit are predominantly inaccessible. We identify 28 B-cell epitopes in the Spike structure and group them as nonaffected by the glycan cloud versus those which are strongly masked by the glycan cloud, resulting in a list of favourable epitopes as targets for vaccine development, antibody-based therapy and diagnostics.

show abstract

Section: N-glycosylation Sites In the Sars-cov-2 S-proteinmentioning

confidence: 99%

Impact of glycan cloud on the B-cell epitope prediction of SARS-CoV-2 Spike protein

Wintjens

Bifani

2020

npj Vaccines

View full text Add to dashboard Cite

show abstract

“…Considering that our main interest is related to glycoprotein hormones, we did not focus other important recombinant protein synthesis still based on CHO or HEK293 cell culture. We just recall some important works based on human cell lines in general (Fliedl et al 2015; Mortazavi et al 2019; Zhong et al 2019), on CHO cell (Bohm et al 2015; Croset et al 2012; Durocher and Butler 2009; Zhang et al 2010) and on HEK293 (Bohm et al 2015; Bouvette et al 2018; Croset et al 2012; Ding et al 2017; Gugliotta et al 2017; Hu et al 2018; Swiech et al 2012; Zhang et al 2010).…”

Section: Discussionmentioning

confidence: 99%

Expression of glycosylated human prolactin in HEK293 cells and related N-glycan composition analysis

et al. 2019

View full text Add to dashboard Cite

Prolactin (PRL) is a hormone produced by the pituitary gland with innumerable functions, such as lactation, reproduction, osmotic and immune regulation. The present work describes the synthesis of hPRL in human embryonic kidney (HEK293) cells, transiently transfected with the pcDNA-3.4-TOPO ® vector carrying the hPRL cDNA. A concentration of ~ 20 mg/L, including glycosylated (G-hPRL) and non-glycosylated (NG-hPRL) human prolactin, was obtained, with ~ 19% of G-hPRL, which is higher than that observed in CHO-derived hPRL (~ 10%) and falling within the wide range of 5–30% reported for pituitary-derived hPRL. N-Glycoprofiling analysis of G-hPRL provided: (i) identification of each N-glycan structure and relative intensity; (ii) average N-glycan mass; (iii) molecular mass of the whole glycoprotein and relative carbohydrate mass fraction; (iv) mass fraction of each monosaccharide. The data obtained were compared to pituitary- and CHO-derived G-hPRL. The whole MM of HEK-derived G-hPRL, determined via MALDI–TOF-MS, was 25,123 Da, which is 0.88% higher than pit- and 0.61% higher than CHO-derived G-hPRL. The main difference with the latter was due to sialylation, which was ~ sevenfold lower, but slightly higher than that observed in native G-hPRL. The “in vitro” bioactivity of HEK-G-hPRL was ~ fourfold lower than that of native G-hPRL, with which it had in common also the number of N-glycan structures.

show abstract

“…Due to extensive post‐translational glycosylation, the hormone exists in a variety of isoforms. For the release of native N‐glycans, rhFSH samples were subjected to enzymatic digestion with PNGase F. These glycans were purified and labeled with the fluorophore 2‐aminobenzamide (2‐AB) as indicated in Gugliotta et al Follitropin CRS standard was processed at the same time and in the same way that the sample solution.…”

Section: Methodsmentioning

confidence: 99%

Recombinant human follicle stimulating hormone purification by a short peptide affinity chromatography

Messina

Giudicessi

Ceron

et al. 2018

Journal of Peptide Science

Self Cite

View full text Add to dashboard Cite

Peptide KVPLITVSKAK was selected to design a synthetic ligand for affinity chromatography purification of recombinant human follicle stimulating hormone (rhFSH), based on the interaction of the hormone with the exoloop 3 of its receptor. The peptide was acetylated to improve its stability to degradation by exopeptidases. A cysteine was incorporated at the C‐termini to facilitate its immobilization to the chromatographic activated SulfoLink agarose resin. A sample of crude rhFSH was loaded to the affinity column, using 20 mM sodium phosphate, 0.5 mM methionine, and pH 5.6 and 7.2 as adsorption and elution buffers, respectively. The dynamic capacity of the matrix was 54.6 mg rhFSH/mL matrix and the purity 94%. The percentage of oxidized rhFSH was 3.4%, and that of the free subunits was 1.2%, both in the range established by the European Pharmacopeia, as also were the sialic acid content and the isoforms profile.

show abstract

Glycosylation and antiproliferative activity of hyperglycosylated IFN-α2 potentiate HEK293 cells as biofactories

Cited by 13 publications

References 53 publications

Impact of glycan cloud on the B-cell epitope prediction of SARS-CoV-2 Spike protein

Impact of glycan cloud on the B-cell epitope prediction of SARS-CoV-2 Spike protein

Expression of glycosylated human prolactin in HEK293 cells and related N-glycan composition analysis

Recombinant human follicle stimulating hormone purification by a short peptide affinity chromatography

Contact Info

Product

Resources

About