Solubilization and refolding of bacterial inclusion body proteins

Singh, Surinder; Panda, Amulya K.

doi:10.1263/jbb.99.303

Cited by 648 publications

(430 citation statements)

References 67 publications

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“…The formation of inclusion bodies could therefore result either from accumulation of high concentrations of folding intermediates or from inefficient processing by molecular chaperones [23]. Refolding from inclusion bodies is in many cases considered undesirable as it results most of the time in a poor recovery of bio-active protein [24]. Most used strategies for shifting the recombinant expression from inclusion bodies to soluble protein are: changing the expression temperature, media and transcription rates, trying various E.coli strains, including molecular chaperones, including tRNA complementation plasmids, expressing fragments, using fusion technology, stabilizing mRNA or screening for soluble variants.…”

Section: Recombinant Expression In Escherichia Colimentioning

confidence: 99%

Exploring three different expression systems for recombinant expression of globins: Escherichia coli, Pichia pastoris and Spodoptera frugiperda

Bracke

Hoogewijs

Dewilde

2018

Analytical Biochemistry

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A B S T R A C TGlobins are among the best investigated proteins in biological and medical sciences and represent a prime tool for the study of the evolution of genes and the structure-function relationship of proteins. Here, we explore the recombinant expression of globins in three different expression systems: Escherichia coli, Pichia pastoris and the baculovirus infected Spodoptera frugiperda. We expressed two different human globin types in these three expression systems: I) the well-characterized neuroglobin and II) the uncharacterized, circular permutated globin domain of the large chimeric globin androglobin. It is clear from the literature that E.coli is the most used expression system for expression and purification of recombinant globins. However, the major disadvantage of E. coli is the formation of insoluble aggregates. We experienced that, for more complex multi-domain globins, like the chimeric globin androglobin, it is recommended to switch to a higher eukaryotic expression system.

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Section: Recombinant Expression In Escherichia Colimentioning

confidence: 99%

Exploring three different expression systems for recombinant expression of globins: Escherichia coli, Pichia pastoris and Spodoptera frugiperda

Bracke

Hoogewijs

Dewilde

2018

Analytical Biochemistry

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“…It also has very strong reversible binding attributes allowing for rapid single-step purification. Polyhistidine tags can be attached on either the N-or C-termini of recombinant proteins, but the optimal location depends on the folding and biochemical characteristics of the adjacent recombinant protein [14,20,26,27] .…”

Section: Refolding Of Solubilized and Unfolded Proteinsmentioning

confidence: 99%

Strategies for production of active eukaryotic proteins in bacterial expression system

Khow

Suntrarachun

2012

Asian Pacific Journal of Tropical Biomedicine

112

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“…The peptide accumulates in inactive aggregates, inclusion bodies (IB), that must be isolated and then solubilized [23]. Structurally, rhIFN-γ monomers have highly hydrophobic domains and contain no disulfide bonds.…”

Section: Introductionmentioning

confidence: 99%

Increased yield of high purity recombinant human interferon-γ utilizing reversed phase column chromatography

Reddy

Narala

et al. 2007

Protein Expression and Purification

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Increasing therapeutic applications for recombinant human interferon-γ (rhIFN-γ), an antiviral proinflammatory cytokine, has broadened interest in optimizing methods for its production and purification. We describe a reversed phase chromatography (RPC) procedure using Source-30 ™ matrix in the purification of rhIFN-γ from Escherichia coli that results in a higher yield than previously reported. The purified rhIFN-γ monomer from the RPC column is refolded in Tris buffer. Optimal refolding occurs at protein concentrations between 50-100 μg/ml. This method yields greater than 90% of the dimer form with a yield of 40 mg g −1 cell mass. Greater than 99% purity is achieved with further purification over a Superdex G-75 column to obtain specific activities of from 2 to 4 × 10 7 IU/mg protein as determined via cytopathic antiviral assay. The improved yield of rhIFN-γ in a simple chromatographic purification procedure promises to enhance the development and therapeutic application of this biologically potent molecule.

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Solubilization and refolding of bacterial inclusion body proteins

Cited by 648 publications

References 67 publications

Exploring three different expression systems for recombinant expression of globins: Escherichia coli, Pichia pastoris and Spodoptera frugiperda

Exploring three different expression systems for recombinant expression of globins: Escherichia coli, Pichia pastoris and Spodoptera frugiperda

Strategies for production of active eukaryotic proteins in bacterial expression system

Increased yield of high purity recombinant human interferon-γ utilizing reversed phase column chromatography

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