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

Zaremba-Czogalla, Magdalena; Stumpp, Christian; Bonifacio, Ezio; Paul, R.

doi:10.1016/j.pep.2015.02.013

Cited by 4 publications

(4 citation statements)

References 41 publications

(47 reference statements)

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“…The laboratory-produced recombinant hIL-7 supported 2E8 cell growth at a protein concentration of as low as 1 ng/mL, which was also a recommended concentration for the commercial standard. A similar optimal concentration was also reported in earlier studies (Cosenza et al 1997;Zaremba-Czogalla et al 2015).…”

Section: Discussionsupporting

confidence: 89%

“…d Growth-promoting activity of purified hIL-7 by cell counting experiment and memory T cells (Mackall et al 2011). In previous reports, a poor expression of hIL-7 in the range of 5-79 mg/L was obtained in the form of IBs in E. coli (Wickham and Walsh 2007;Zaremba-Czogalla et al 2015). Therefore, in this study, we have addressed the problem of its poor expression via optimization of host-vector combinations, where different expression constructs were designed using native and codonoptimized hIL-7 gene to improve its production yields.…”

Section: Discussionmentioning

confidence: 98%

“…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). 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: 98%

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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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Section: Discussionsupporting

confidence: 89%

Section: Discussionmentioning

confidence: 98%

Section: Introductionmentioning

confidence: 98%

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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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“…Очистка IL-7 перед рефолдингом давала змогу отримати більшу кількість мономерного та біологічно активного IL-7. Крім того, додаткова стадія очистки до рефолдингу дає змогу знизити вміст ендотоксинів у кінцевому білковому препараті [45].…”

Section: виділення та очистка рекомбінантних цитокінівunclassified

Comparative Characteristics of Biotechnological Approaches to Obtaining Recombinant Human Cytokines in Bacterial Expressing Systems

Natochii¹,

Motronenko²

2019

Innov Biosyst Bioeng

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Background. Cytokines provide a link between cells and play an important role in the innate and adaptive immune response modulation. As mediators, they are active in very small amounts and effect many biological processes: embryonic development, nonspecific and specific immune responses, changes in cognitive function, to name a few. Recombinant microorganisms are commonly used to produce cytokine preparations. Perspectives of therapeutic use of recombinant cytokines make it urgent to create new and improve existing technologies for their biosynthesis, isolation and purification. Objective. The purpose of the paper is analysis and comparison of biotechnological approaches for the production of human recombinant cytokines, based on current literature, and identifying promising ways to improve the efficiency of their production technologies. Methods. The analysis and systematization of modern scientific works devoted to the production of recombinant cytokines with determination of peculiarities of the course of their biosynthesis process and to carry out comparative characteristics of different producers. Results. Most often, to obtain recombinant cytokines, Escherichia coli based expression systems are used, as they are the best-studied to allow high yield of the target product. Their biosynthesis, as a rule, is carried out by a method of periodic fermentation with nutrition, and to the composition of the nutrient medium can be added stimulants of biosynthesis of different origin. Bacterial producers have several disadvantages: the accumulation of the target protein is most often in the form of Taurus inclusions, and the question remains of contamination of such drugs with bacterial endotoxins. To negate these drawbacks, they resort to optimizing the procedures for isolating and purifying recombinant proteins. Conclusions. One of the promising areas of current research on the production of recombinant cytokines is the various technologies for obtaining the target protein in soluble form. However, the complexity of this task lies in the lack of the ability to create a universal method and requires an individual approach, depending on the producer and the final product. Also promising are studies to increase the yield of the target protein by changing the culture conditions and composition of the nutrient medium, eliminating endotoxin infection, and finding alternative bacterial expression systems.

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Efficient expression of a cytokine combination in Saccharomyces cerevisiae for cultured meat production

Lei

et al. 2023

Food Research International

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

Cited by 4 publications

References 41 publications

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

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

Comparative Characteristics of Biotechnological Approaches to Obtaining Recombinant Human Cytokines in Bacterial Expressing Systems

Efficient expression of a cytokine combination in Saccharomyces cerevisiae for cultured meat production

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