Optimization of refolding with simultaneous purification of recombinant human granulocyte colony-stimulating factor from Escherichia coli by immobilized metal ion affinity chromatography

Wang, Chaozhan; Wang, Lili; Geng, Xindu

doi:10.1016/j.bej.2008.09.018

Cited by 33 publications

(11 citation statements)

References 35 publications

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“…PFLC has been applied successfully for the renaturation and simultaneous purification of some recombinant proteins produced by E. coli, such as recombinant human interferon-γ (rhINF-γ) (18), recombinant human proinsulin (19), recombinant human granulocyte colony-stimulating factor (rhG-CSF) (20), and recombinant human stem cell factor (rhSCF) (21), and the recovery of bioactivity of these renatured recombinant proteins was increased 2-3 times than that of other usual methods.…”

Section: Introductionmentioning

confidence: 99%

Renaturation and Purification of rhGM-CSF with Ion-Exchange Chromatography

Bai

Chen

et al. 2007

Biotechnol. Prog.

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The renaturation and purification of recombinant human granulocyte macrophage colony stimulation factor (rhGM-CSF) expressed in Escherichia coli with strong anion-exchange chromatography (SAX) were studied. The effects of pH values, ratios of concentrations of GSH/GSSG, and urea concentrations in the mobile phase on the renaturation and purification of rhGM-CSF with SAX were investigated, respectively. The results show that the above three factors have remarkable influences on the efficiency of renaturation and mass recovery of rhGM-CSF. The addition of GSH/GSSG in the mobile phase can improve the formation of correct disulfide bonds in rhGM-CSF so that its renaturation yield increases. In addition, to enhance the mass recovery of rhGM-CSF with SAX, the low concentration of urea was added in the mobile phase to prevent denatured protein aggregation. Under the optimal conditions, rhGM-CSF was renatured with simultaneous purification on SAX column within 30 min only by one step. After that its specific bioactivity, mass recovery, and purity reached 1.66 x 10(7) IU x mg, 58.8%, and 96.2%, respectively.

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Section: Introductionmentioning

confidence: 99%

Renaturation and Purification of rhGM-CSF with Ion-Exchange Chromatography

Bai

Chen

et al. 2007

Biotechnol. Prog.

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“…The recombinant plasmid, pTrcHis flbS ∗ , was transformed into and expressed in E. coli DH5α. His6‐tagged FlbS ∗ was purified from the inclusion bodies as described by the manufacturer but by using a column renaturation step [12].…”

Section: Methodsmentioning

confidence: 99%

FixL‐like sensor FlbS of Brucella abortus binds haem and is necessary for survival within eukaryotic cells

Roset

Almirón

2013

FEBS Letters

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Edited by Renee TsolisKeywords: FixL Brucella Haemoprotein Intracellular survival Two-component system a b s t r a c t Replication of Brucella inside eukaryotic cells is essential for pathogenesis, and successful infection requires rapid adaptation to the intracellular milieu. Close relatives of Brucella use the two-component system FixLJ to survive inside the host. We aimed to identify a homologous sensor in Brucella abortus. A predicted protein with transmembrane and conserved histidine kinase domains was identified as the Fix-like Brucella sensor, FlbS. Although it lacks the PAS domain, recombinant FlbS binds haem in vitro. An internal in-frame deletion in flbS severely decreased B. abortus survival inside professional and non-professional phagocytes. This phenotype was reverted by genetic complementation. These results indicate the critical role of this haemoprotein in the intracellular lifestyle of Brucella.

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“…Previously we refolded rhG-CSF, which was solubilized by 8 M urea, by using various liquid chromatographic methods [13,[23][24][25][26]. Though higher refolding yields were approached when compared to dilution refolding method, the highest mass recovery in these work is only 49% [24].…”

Section: Refolding Of Rhg-csf By Using Hicmentioning

confidence: 99%

“…When rhG-CSF is produced in E. coli, it often forms inclusion bodies, thus solubilization and subsequent refolding are necessary to recover the useful and bioactive protein. We have extensively investigated the refolding and purification of urea-solubilized rhG-CSF from inclusion bodies by using various chromatography-based methods [13,[23][24][25][26]. However, the protein yield is still anticipated to be enhanced.…”

Section: Introductionmentioning

confidence: 99%

High pH Solubilization and Chromatography-Based Renaturation and Purification of Recombinant Human Granulocyte Colony-Stimulating Factor from Inclusion Bodies

Fan

Liu

et al. 2012

Appl Biochem Biotechnol

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Recombinant human granulocyte colony-stimulating factor (rhG-CSF) is a very efficient therapeutic protein drug which has been widely used in human clinics to treat cancer patients suffering from chemotherapy-induced neutropenia. In this study, rhG-CSF was solubilized from inclusion bodies by using a high-pH solution containing low concentration of urea. It was found that solubilization of the rhG-CSF inclusion bodies greatly depended on the buffer pH employed; alkalic pH significantly favored the solubilization. In addition, when small amount of urea was added to the solution at high pH, the solubilization was further enhanced. After solubilization, the rhG-CSF was renatured with simultaneous purification by using weak anion exchange, strong anion exchange, and hydrophobic interaction chromatography, separately. The results indicated that the rhG-CSF solubilized by the high-pH solution containing low concentration of urea had much higher mass recovery than the one solubilized by 8 M urea when using anyone of the three refolding methods employed in this work. In the case of weak anion exchange chromatography, the high pH solubilized rhG-CSF could get a mass recovery of 73%. The strategy of combining solubilization of inclusion bodies at high pH with refolding of protein using liquid chromatography may become a routine method for protein production from inclusion bodies.

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Optimization of refolding with simultaneous purification of recombinant human granulocyte colony-stimulating factor from Escherichia coli by immobilized metal ion affinity chromatography

Cited by 33 publications

References 35 publications

Renaturation and Purification of rhGM-CSF with Ion-Exchange Chromatography

Renaturation and Purification of rhGM-CSF with Ion-Exchange Chromatography

FixL‐like sensor FlbS of Brucella abortus binds haem and is necessary for survival within eukaryotic cells

High pH Solubilization and Chromatography-Based Renaturation and Purification of Recombinant Human Granulocyte Colony-Stimulating Factor from Inclusion Bodies

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