Expression, purification, and functional characterization of recombinant human interleukin-7

Luo, Yong Ming; Kong, Xiangping; Xu, Aimin; Jin, Song; Wu, Donghai

doi:10.1016/j.pep.2008.08.009

Cited by 14 publications

(10 citation statements)

References 21 publications

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“…Dot blot analysis with an IL-7 specific polyclonal antibody revealed that the three X33/IL7 clones secreted in a higher amount compared to three GS115/IL7 clones, and that the maximum amount of IL-7 was present between 72 h and 120 h after the start of methanol induction ( Supplementary Fig. S1), in accordance with the findings of Luo et al [20].…”

Section: Il-7 Expression With P Pastorissupporting

confidence: 89%

“…It was previously reported that P. pastoris can secrete recombinantly expressed IL-7 that can be purified from the growth medium by cation exchange chromatography [20]. As P. pastoris clones are selected on agar plates, we reasoned that it would be possible to generate a library encoding IL-7 mutant proteins in P. pastoris, which after secretion into and purification from the growth medium could be screened for activity.…”

Section: Il-7 Expression With P Pastorismentioning

confidence: 99%

“…We first tested expression of IL-7 with the eukaryotic yeast Pichia pastoris, which was reported to secrete folded IL-7 into the growth medium [20]. We found that Pichiaexpressed IL-7 was active, but highly N-linked glycosylated, resulting in an apparent molecular weight of $40-80 kDa.…”

Section: Introductionmentioning

confidence: 99%

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Comparison of the purification of biologically active IL-7 cytokine expressed in Escherichia coli and Pichia pastoris

Zaremba-Czogalla

Stumpp

Bonifacio

et al. 2015

Protein Expression and Purification

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Section: Il-7 Expression With P Pastorissupporting

confidence: 89%

Section: Il-7 Expression With P Pastorismentioning

confidence: 99%

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Comparison of the purification of biologically active IL-7 cytokine expressed in Escherichia coli and Pichia pastoris

Zaremba-Czogalla

Stumpp

Bonifacio

et al. 2015

Protein Expression and Purification

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“…The nonglycosylated rhIL-7 produced in E. coli, although bioactive, may induce immunogenicity issue, via repeated injections and/or at high-dose regimen. rhIL-7 has also been produced in Pichia pastoris [15], but glycosylation pattern was shown to be different to that typically found in human. Very recently, Mirzaei et al, have reported production and purification of rhIL-7 in insect cells using the baculovirus expression vector [16].…”

Section: Introductionmentioning

confidence: 93%

CZE for glycoform profiling and quality assessment of recombinant human interleukin‐7

Alahmad

Tran

Duboeuf³

et al. 2009

Electrophoresis

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The aim of the present work was to develop a simple high-resolution CZE method for glycoform profiling and quality assessment of recombinant human interleukin-7 (rhIL-7) produced in Chinese hamster ovary cells. Classical and isoelectric buffers and several monoamines used as alkaline component of BGE at very low concentration have been investigated in order to optimize the resolution. The best separation was achieved using a fused-silica capillary and a buffer composed of 25 mM citrate/triethanolamine at pH 2.6. Under these acidic conditions, triethanolamine was able to reverse EOF favorably and to limit rhIL-7 adsorption. This method allowed the separation of four groups of peaks ranging from low to high-sialylated glycoforms. An extensive study on inter-run rinsing procedures has been conducted. Rinsing with 50 mM SDS was retained to achieve the optimal repeatability. Excellent intermediate precision was obtained for migration time (RSD < 0.6%), while RSD for intraday studies were only less than 2.9%. Satisfactory inter and intraday repeatabilities were also observed for relative peak area. We finally demonstrated that reliable information could be obtained to address comparability studies and demonstrate batch-to-batch consistency of biomanufactured rhIL-7.

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“…The heterologous expression of hIL-7 protein has been tested in different hosts such as Escherichia coli (Cosenza et al 1997), Pichia pastoris (35 mg/L) (Luo et al 2009), and baculovirus (124 mg/L) (Mirzaei et al 2008). The expression level in E. coli hosts was uniformly poor in the range of 5-79 mg/L (Wickham and Walsh 2007;Zaremba-Czogalla et al 2015).…”

Section: Introductionmentioning

confidence: 99%

A combinatorial approach of N-terminus blocking and codon optimization strategies to enhance the soluble expression of recombinant hIL-7 in E. coli fed-batch culture

Devi

Adivitiya

Khasa

2016

Appl Microbiol Biotechnol

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Human interleukin-7 (hIL-7) is a therapeutically important cytokine involved in lymphocyte development and survival. In previous reports, a uniformly poor expression of hIL-7 has been shown in Escherichia coli host with the problem of inclusion body formation. In this study, the role of codon optimization and N-terminus blocking using various solubility enhancer fusion tags was explored to improve its soluble expression. The use of codon optimization strategy improved its expression to 80 ± 5 mg/L at shake flask level. The utilization of pelB leader sequence resulted in an unprocessed protein in the form of cytoplasmic inclusion bodies with lower expression yields. The N-terminus fusion of small ubiquitin-like modifier (SUMO), thioredoxin (Trx), and NusA tags increased the expression in the range of 90-140 mg/L, where >90 % of the fusion protein was obtained in soluble form. The fed-batch fermentation of SUMO-tagged hIL-7 protein was optimized at bioreactor level, where a high volumetric product concentration of 2.65 g/L was achieved by controlling the plasmid segregation instability using high antibiotic concentration. The specific product yield (Y) and volumetric product concentration were 1.38 and 2.55-fold higher compared to batch results, respectively. A preparative scale affinity chromatography resulted in a high recovery yield of 50.6 mg/L with ∼90 % purity. The conformational property of purified recombinant hIL-7 from CD spectroscopy showed a typical helical structure with 31.5 % α-helix and 26.43 % β-sheet. The biological activity of purified protein was tested using IL-7-dependent murine immature B lymphocyte (2E8) cell line by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide salt (MTT) assay, where it showed a similar biological activity as standard control.

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Expression, purification, and functional characterization of recombinant human interleukin-7

Cited by 14 publications

References 21 publications

Comparison of the purification of biologically active IL-7 cytokine expressed in Escherichia coli and Pichia pastoris

Comparison of the purification of biologically active IL-7 cytokine expressed in Escherichia coli and Pichia pastoris

CZE for glycoform profiling and quality assessment of recombinant human interleukin‐7

A combinatorial approach of N-terminus blocking and codon optimization strategies to enhance the soluble expression of recombinant hIL-7 in E. coli fed-batch culture

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