Production and purification of refolded recombinant human IL-7 from inclusion bodies

Ouellette, Thomas; Destrau, Sophie; Ouellette, Timothy F.; Zhu, Jianwei; Roach, John A. G.; Coffman, J.Daniel; Hecht, Toby T.; Lynch, James; Giardina, Steven L.

doi:10.1016/s1046-5928(03)00134-7

Cited by 30 publications

(16 citation statements)

References 26 publications

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“…Finally, when captured in inclusion bodies, the recombinant protein is considered to be protected against proteases [26,150,165]. Consequently, this strategy has been applied in numerous examples [6,79,87,122,164]. Main drawbacks remain the absence of generally applicable and inexpensive methods to refold the protein and the low overall yields of downstream processing [150].…”

Section: Optimization Of Inclusion Body Formationmentioning

confidence: 99%

Increasing recombinant protein production in Escherichia coli through metabolic and genetic engineering

Waegeman¹,

Soetaert²

2011

J Ind Microbiol Biotechnol

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Different hosts have been used for recombinant protein production, ranging from simple bacteria, such as Escherichia coli and Bacillus subtilis, to more advanced eukaryotes as Saccharomyces cerevisiae and Pichia pastoris, to very complex insect and animal cells. All have their advantages and drawbacks and not one seems to be the perfect host for all purposes. In this review we compare the characteristics of all hosts used in commercial applications of recombinant protein production, both in the area of biopharmaceuticals and industrial enzymes. Although the bacterium E. coli remains a very often used organism, several drawbacks limit its possibility to be the first-choice host. Furthermore, we show what E. coli strains are typically used in high cell density cultivations and compare their genetic and physiological differences. In addition, we summarize the research efforts that have been done to improve yields of heterologous protein in E. coli, to reduce acetate formation, to secrete the recombinant protein into the periplasm or extracellular milieu, and to perform post-translational modifications. We conclude that great progress has been made in the incorporation of eukaryotic features into E. coli, which might allow the bacterium to regain its first-choice status, on the condition that these research efforts continue to gain momentum.

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Section: Optimization Of Inclusion Body Formationmentioning

confidence: 99%

Increasing recombinant protein production in Escherichia coli through metabolic and genetic engineering

Waegeman¹,

Soetaert²

2011

J Ind Microbiol Biotechnol

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“…In this work, the high recovery yield of soluble hIL-7 protein using affinity chromatography from fusion partner was strongly favored over literature reports where its retrieval from inclusion bodies resulted in a poor yield in the range of 5-10 mg/L (Ouellette et al 2003). Moreover, this strategy is expected to result in an authentic N-terminus of the fusion partner due to the high sequence specificity of SUMO protease (Butt et al 2005).…”

Section: Discussionmentioning

confidence: 99%

“…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). The maximum hIL-7 expression achieved even in the bioreactor (720 L) was at a level of 10 mg/L after optimization of batch fermentation conditions (Ouellette et al 2003).…”

Section: Introductionmentioning

confidence: 97%

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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“…[11,12], several expression systems have been developed to produce recombinant rhIL-7. Currently, rhIL-7 is mainly produced in Escherichia coli expression system, which involves accumulation in inclusion bodies [13,14]. Although high expression levels are obtained in the bacterium, purification from inclusion bodies is usually quite difficult and leads to poor yields of refolded bioactive pharmaceutical ingredient.…”

Section: Introductionmentioning

confidence: 99%

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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Production and purification of refolded recombinant human IL-7 from inclusion bodies

Cited by 30 publications

References 26 publications

Increasing recombinant protein production in Escherichia coli through metabolic and genetic engineering

Increasing recombinant protein production in Escherichia coli through metabolic and genetic engineering

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

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

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