Refolding of therapeutic proteins produced inEscherichia coli as inclusion bodies

Misawa, S.; Kumagai, Izumi

doi:10.1002/(sici)1097-0282(1999)51:4<297::aid-bip5>3.0.co;2-i

Cited by 147 publications

(68 citation statements)

References 83 publications

(89 reference statements)

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“…The chaotropic properties of urea make it useful as a strong denaturant. It has seen broad application for the solubilization and refolding of protein inclusion bodies obtained from Escherichia coli (e.g., [1][2][3][4][5][6]). There have been a smaller number of reports of urea used in conjunction with ion-exchange adsorption.…”

Section: Introductionmentioning

confidence: 99%

Impact of denaturation with urea on recombinant apolipoprotein A‐I_Milano ion‐exchange adsorption: Equilibrium uptake behavior and protein mass transfer kinetics

Hunter

Suda²,

Das

et al. 2007

Biotechnology Journal

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We have studied the equilibrium uptake behavior and mass transfer rate of recombinant apolipoprotein A-I Milano (apo A-I M ) on Q Sepharose HP under non-denaturing, partially denaturing, and fully denaturing conditions. The protein of interest in this study is composed of amphipathic α helices that serve to solubilize and transport lipids. The dual nature of this molecule leads to the formation of micellar-like structures and self association in solution. Under non-denaturing conditions equilibrium uptake is 134 mg/mL media and the isotherm is essentially rectangular. When fully denatured with 6 M urea, the equilibrium binding capacity decreases to 25 mg/mL media and the isotherm becomes less favorable. The decrease in both binding affinity and media capacity when the protein is completely denatured with 6 M urea can be explained by the loss of all alpha helical structure. The rate of apo A-I M mass transfer on Q Sepharose HP was characterized using a macropore diffusion model. Results of modeling studies indicate that effective pore diffusivity increases from 4.5 × 10 -9 cm 2 /s in the absence of urea to 6.0 × 10 -8 cm 2 /s when apo A-I M is fully denatured with 6 M urea. Based on light-scattering data reported for apo A-I, protein self association appears to be the dominant cause of slow protein mass transfer observed under non-denaturing conditions.

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Section: Introductionmentioning

confidence: 99%

Impact of denaturation with urea on recombinant apolipoprotein A‐I_Milano ion‐exchange adsorption: Equilibrium uptake behavior and protein mass transfer kinetics

Hunter

Suda²,

Das

et al. 2007

Biotechnology Journal

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“…[3][4][5][6][7] Proteins expressed as inclusion bodies were denatured, purified, and refolded to yield soluble forms. 8,9 Several proteins showed enhanced solubility and long-term stability by simultaneous addition of charged amino acids L-Arg and L-Glu at 50 mM to the buffer, which increased the maximal concentration up to 8.7 times.…”

Section: 4mentioning

confidence: 99%

Solution Structure of Water-soluble Mutant of Crambin and Implication for Protein Solubility

Kang¹,

Lim²,

Lee³

et al. 2011

Bulletin of the Korean Chemical Society

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Water-soluble mutant of intrinsically insoluble protein, crambin, was produced by mutagenesis based on the sequence analysis with homologous proteins. Thr1, Phe13, and Lys33 of crambin were substituted for Lys, Tyr, and Lys, respectively. The resultant mutant was soluble in aqueous buffer as well as in dodecylphosphocholine (DPC) micelle solution. The H- 15N spectrum of the mutant crambin showed spectral similarity to that of the wild-type protein except for local regions proximal to the sites of mutation. Solution structure of water-soluble mutant crambin was determined in aqueous buffer by NMR spectroscopy. The structure was almost identical to the wild-type structure determined in non-aqueous solvent. Subtle difference in structure was very local and related to the change of the intra-and inter-protein hydrophobic interaction of crambin. The structural details for the enhanced solubility of crambin in aqueous solvent by the mutation were provided and discussed.

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“…Refolding of denatured Trx-r-PA with different post-treatments Refolding by dilution The five samples mentioned above were respectively diluted into refolding buffer A (Table 1) by a dilution factor of 21 (Misawa & Kumagai 1999).…”

Section: Instruments and Materialsmentioning

confidence: 99%

A Comparative Investigation on Different Refolding Strategies of Recombinant Human Tissue-type Plasminogen Activator Derivative

2006

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Recombinant human tissue-type plasminogen activator derivative (r-PA), fused with thioredoxin (Trx), was expressed in Escherichia coli. The resultant fusion protein, Trx-r-PA, was almost completely in the form of inclusion bodies and without activity. Different refolding strategies were investigated including different post-treatment of solubilized Trx-r-PA inclusion bodies, on-column refolding by size-exclusion chromatography (SEC) using three gel types (Sephacryl S-200, S-300 and S-400), refolding by Sephacryl S-200 with a urea gradient and two-stage temperature control in refolding. An optimized on-column refolding process for Trx-r-PA inclusion bodies was established. The collected Trx-r-PA inclusion bodies were dissolved in 6 M: guanidine hydrochloride (Gdm.HCl), and the denatured protein was separated from dithiothreitol (DTT) and Gdm.HCl with a G25 column and simultaneously dissolved in 8 M: urea containing oxidized glutathione (GSSG). Finally a refolding of Trx-r-PA protein on Sephacryl S-200 column with a decreasing urea gradient combined with two-stage temperature control was employed, and the activity recovery of refolded protein was increased from 3.6 to 13.8% in comparison with the usual dilution refolding.

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Refolding of therapeutic proteins produced inEscherichia coli as inclusion bodies

Cited by 147 publications

References 83 publications

Impact of denaturation with urea on recombinant apolipoprotein A‐I_Milano ion‐exchange adsorption: Equilibrium uptake behavior and protein mass transfer kinetics

Impact of denaturation with urea on recombinant apolipoprotein A‐I_Milano ion‐exchange adsorption: Equilibrium uptake behavior and protein mass transfer kinetics

Solution Structure of Water-soluble Mutant of Crambin and Implication for Protein Solubility

A Comparative Investigation on Different Refolding Strategies of Recombinant Human Tissue-type Plasminogen Activator Derivative

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Refolding of therapeutic proteins produced inEscherichia coli as inclusion bodies

Cited by 147 publications

References 83 publications

Impact of denaturation with urea on recombinant apolipoprotein A‐IMilano ion‐exchange adsorption: Equilibrium uptake behavior and protein mass transfer kinetics

Impact of denaturation with urea on recombinant apolipoprotein A‐IMilano ion‐exchange adsorption: Equilibrium uptake behavior and protein mass transfer kinetics

Solution Structure of Water-soluble Mutant of Crambin and Implication for Protein Solubility

A Comparative Investigation on Different Refolding Strategies of Recombinant Human Tissue-type Plasminogen Activator Derivative

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Impact of denaturation with urea on recombinant apolipoprotein A‐I_Milano ion‐exchange adsorption: Equilibrium uptake behavior and protein mass transfer kinetics

Impact of denaturation with urea on recombinant apolipoprotein A‐I_Milano ion‐exchange adsorption: Equilibrium uptake behavior and protein mass transfer kinetics