Advances in refolding of proteins produced in E. coli

Lilie, Hauke; Schwarz, Elisabeth; Rudolph, Rainer

doi:10.1016/s0958-1669(98)80035-9

Cited by 414 publications

(230 citation statements)

References 35 publications

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“…Native EGF contains three disulfide bonds that are vital for its biological activity. Since the reducing environment of the bacterial cytosol prevents the formation of these disulfide bonds, it promotes the manifestation of protein aggregates in these IBs [20]. Therefore, the EGF recovered from these IBs requires refolding in order to regain its biological function.…”

Section: Discussionmentioning

confidence: 99%

Escherichia coli expression and refolding of E/K-coil-tagged EGF generates fully bioactive EGF for diverse applications

Lenferink

Pinard

et al. 2009

Protein Expression and Purification

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Section: Discussionmentioning

confidence: 99%

Escherichia coli expression and refolding of E/K-coil-tagged EGF generates fully bioactive EGF for diverse applications

Lenferink

Pinard

et al. 2009

Protein Expression and Purification

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“…However, the inclusion body protein has to be solubilized, purified, and properly folded in membranes or detergent. A typical protocol includes concomitant denaturation and solubilization using a solution containing a denaturant such as urea, purification, and then refolding with solutions containing detergent and possibly lipid vesicles [5][6][7]. In this paper, refolding was achieved by first mixing the solution of purified denatured fusion protein with a solution containing high concentration detergent and then dialyzing to reduce the denaturant concentration to a negligible level.…”

Section: Introductionmentioning

confidence: 99%

Isotopically labeled expression in E. coli, purification, and refolding of the full ectodomain of the influenza virus membrane fusion protein

Curtis-Fisk

Spencer

Weliky

2008

Protein Expression and Purification

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This paper describes methods to produce an isotopically labeled 23 kDa viral membrane protein with purified yield of 20 mg/L of E. coli shake flask culture. This yield is sufficient for NMR structural studies and the protein production methods are simple, straightforward, and rapid and likely applicable to other recombinant membrane proteins expressed in E. coli. The target FHA2 protein is the full ectodomain construct of the influenza virus hemagglutinin protein which catalyzes fusion between the viral and the cellular endosomal membranes during infection. The high yield of FHA2 was achieved by: (1) initial growth in rich medium to A 600 ~ 8 followed by a switch to minimal medium and induction of protein expression; and (2) obtaining protein both from purification of the detergent-soluble lysate and from solubilization, purification, and refolding of inclusion bodies. The high cell density was achieved after optimization of pH, oxygenation, and carbon source and concentration, and the refolding protocol was optimized using circular dichroism spectroscopy. For a single residue of membrane-associated FHA2 that was obtained from purification and refolding of inclusion bodies, native conformation was verified by the 13 CO chemical shift measured using solidstate nuclear magnetic resonance spectroscopy.

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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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Advances in refolding of proteins produced in E. coli

Cited by 414 publications

References 35 publications

Escherichia coli expression and refolding of E/K-coil-tagged EGF generates fully bioactive EGF for diverse applications

Escherichia coli expression and refolding of E/K-coil-tagged EGF generates fully bioactive EGF for diverse applications

Isotopically labeled expression in E. coli, purification, and refolding of the full ectodomain of the influenza virus membrane fusion protein

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

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Advances in refolding of proteins produced in E. coli

Cited by 414 publications

References 35 publications

Escherichia coli expression and refolding of E/K-coil-tagged EGF generates fully bioactive EGF for diverse applications

Escherichia coli expression and refolding of E/K-coil-tagged EGF generates fully bioactive EGF for diverse applications

Isotopically labeled expression in E. coli, purification, and refolding of the full ectodomain of the influenza virus membrane fusion protein

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

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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