Purification and characterization of human IL-10/Fc fusion protein expressed in Pichia pastoris

Guo, Yugang; Kang, Wenyao; Zhong, Yaohua; Li, Rui; Li, Guangwei; Shen, Yi; Hu, Shengyong; Sun, Jie; Xiao, Weihua

doi:10.1016/j.pep.2012.03.012

Cited by 12 publications

(4 citation statements)

References 23 publications

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“…As reported previously 51 – 53 , the IL-10/Fc fusion protein containing a human IL-10 fused at the N-terminal with a noncytolytic human IgG1 Fc was expressed by FreeStyle 293-F Cells (Gibco / Thermo Fisher Scientific) at the EPFL Protein Expression Core Facility. Supernatant of culture medium containing IL-10/Fc fusion protein was harvested by centrifugation after a 7-day culture and was filtered through a 0.22-μm membrane to obtain a clear solution.…”

Section: Methodsmentioning

confidence: 99%

Metabolic reprogramming of terminally exhausted CD8+ T cells by IL-10 enhances anti-tumor immunity

et al. 2021

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T cell exhaustion presents one of the major hurdles to cancer immunotherapy. Among exhausted CD8 + tumor-infiltrating lymphocytes (TILs), the terminally exhausted subset contributes directly to tumor cell killing owing to its cytotoxic effector function. However, this subset does not respond to immune checkpoint blockades and is difficult to be reinvigorated with restored proliferative capacity. Here, we show that a half-life–extended interleukin (IL)-10/Fc fusion protein directly and potently enhanced expansion and effector function of terminally exhausted CD8 + TILs by promoting oxidative phosphorylation (OXPHOS), a process independent of the progenitor exhausted T cells. IL-10/Fc was a safe and highly efficient metabolic intervention that synergized with adoptive T cell transfer immunotherapy, leading to eradication of established solid tumors and durable cures in a majority of treated mice. These findings show that metabolic reprogramming by upregulating mitochondrial pyruvate carrier-dependent OXPHOS can revitalize terminally exhausted T cells and enhance the response to cancer immunotherapy.

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Section: Methodsmentioning

confidence: 99%

Metabolic reprogramming of terminally exhausted CD8+ T cells by IL-10 enhances anti-tumor immunity

et al. 2021

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“…The recombinant protein harvested from the P. pastoris MNN9-EndoT strains was then purified by affinity chromatography on a protein G column and approximate 200-250 mg of recombinant IgG1-Fc were obtained from 1 L of fermentation medium (Fig. 3b, Additional file 1: Figure S5), which was higher than the previous reports (from 10 to 100 mg/L) [51][52][53]. The purified IgG1-Fc from WT and MNN9-EndoT strain were detected by ConA blot (Additional file 1: Figure S6), suggesting the truncated N-glycan in engineered strain.…”

Section: Characterization Of Igg1-fc Region With N-glcnacmentioning

confidence: 74%

Homogeneous production and characterization of recombinant N-GlcNAc-protein in Pichia pastoris

et al. 2020

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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.

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“…The linearized vectors were transformed into P. pastoris strain GS115. The positive transformants were screened by MD plates and examined by Western blot using anti-human IgG-HRP conjugates according to a previous report [14]. …”

Section: Methodsmentioning

confidence: 99%

Elimination of N-glycosylation by site mutation further prolongs the half-life of IFN-α/Fc fusion proteins expressed in Pichia pastoris

et al. 2016

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BackgroundInterferon (IFN)-α has been commonly used as an antiviral drug worldwide; however, its short half-life in circulation due to its low molecular weight and sensitivity to proteases impacts its efficacy and patient compliance.ResultsIn this study, we present an IgG1 Fc fusion strategy to improve the circulation half-life of IFN-α. Three different forms of IgG1 Fc fragments, including the wild type, aglycosylated homodimer and aglycosylated single chain, were each fused with IFN-α and designated as IFN-α/Fc-WT, IFN-α/Fc-MD, and IFN-α/Fc-SC, respectively. The recombinant proteins were expressed in Pichia pastoris and tested using antiviral and pharmacokinetic assays in comparison with the commercial pegylated-IFN-α (PEG-IFN-α). The in vitro study demonstrated that IFN-α/Fc-SC has the highest antiviral activity, while IFN-α/Fc-WT and IFN-α/Fc-MD exhibited antiviral activities comparable to that of PEG-IFN-α. The in vivo pharmacokinetic assay showed that both IFN-α/Fc-WT and IFN-α/Fc-MD have a longer half-life than PEG-IFN-α in SD rats, but IFN-α/Fc-SC has the shortest half-life among them. Importantly, the circulating half-life of 68.3 h for IFN-α/Fc-MD was significantly longer than those of 38.2 h for IFN-α/Fc-WT and 22.2 h for PEG-IFN-α.ConclusionsThe results demonstrate that the elimination of N-glycosylation by mutation of putative N-glycosylation site further prolongs the half-life of the IFN-α/Fc fusion protein and could present an alternative strategy for extending the half-life of low-molecular-weight proteins expressed by P. pastoris for in vivo studies as well as for future clinical applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0601-9) contains supplementary material, which is available to authorized users.

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Purification and characterization of human IL-10/Fc fusion protein expressed in Pichia pastoris

Cited by 12 publications

References 23 publications

Metabolic reprogramming of terminally exhausted CD8+ T cells by IL-10 enhances anti-tumor immunity

Metabolic reprogramming of terminally exhausted CD8+ T cells by IL-10 enhances anti-tumor immunity

Homogeneous production and characterization of recombinant N-GlcNAc-protein in Pichia pastoris

Elimination of N-glycosylation by site mutation further prolongs the half-life of IFN-α/Fc fusion proteins expressed in Pichia pastoris

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