Chromatographic Methods for the Isolation of, and Refolding of Proteins from, Escherichia coli Inclusion Bodies

Gu, Zhenyu; Weidenhaupt, Marianne; Ivanovа, Natalia; Pavlov, Michail; Xu, Bingze; Su, Zhiguo; Janson, Jan‐Christer

doi:10.1006/prep.2002.1624

Cited by 71 publications

(43 citation statements)

References 13 publications

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“…2, lane 3 and 8). The dissolved inclusion bodies were then subjected to a Sephadex G-25 column to remove urea gently, which could inhibit protein aggregation, resulting in an enhanced refolding yield (Gu et al 2002). The final product of SMP1 was 43.1 mg from 1 l cell culture, and SMP2 was 49.6 mg.…”

Section: Resultsmentioning

confidence: 99%

Cloning and Expression of Human Stem Cell Factor Fused with Thrombopoietin Mimetic Peptide in Escherichia coli

Chen

Yang

et al. 2006

Biotechnol Lett

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Thrombopoietin (TPO) acts synergistically with stem cell factor (SCF) in hematopoiesis and megakaryopoiesis. In this work, we designed the expression of SCF fused with the monomer or the dimer of TPO mimetic peptide through a flexible peptide linker. The recombinant fusion proteins were produced in E. coli DH5alpha at up to 25% of total cell proteins. The resultant inclusion bodies were refolded by dilution and purified by ion-exchange chromatography. Subsequent biological activity assays showed that the fusion proteins exhibited higher potency than recombinant human SCF, indicating that they have a potential role for pharmaceutical applications.

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Section: Resultsmentioning

confidence: 99%

Cloning and Expression of Human Stem Cell Factor Fused with Thrombopoietin Mimetic Peptide in Escherichia coli

Chen

Yang

et al. 2006

Biotechnol Lett

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“…Moreover, the aggregates formed during dialysis and diafiltration are usually more drastic than that during dilution refolding. 5 An efficient way that promotes refolding and simultaneously minimizes aggregation at high protein concentration will significantly improve the yield of target protein as well as reduce the cost of production. 6 Recent literature data have provided information aimed at enhancing the refolding yield of inclusion body proteins by reducing the causes of aggregation and misfolded configurations, respectively.…”

Section: Introductionmentioning

confidence: 99%

Refolding of denatured/reduced lysozyme at high concentrations by artificial molecular chaperone‐ion exchange chromatography

Wang

Zhang

Chen

et al. 2010

Biotechnology Progress

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Development of high efficiency and low cost protein refolding methods is a highlighted research focus in biotechnology. Artificial molecular chaperone (AMC) and protein folding liquid chromatography (PFLC) are two attractive refolding methods developed in recent years. In the present work, AMC and one branch of PFLC, ion exchange chromatography (IEC), are integrated to form a new refolding method, artificial molecular chaperone-ion exchange chromatography (AMC-IEC). This new method is applied to the refolding of a widely used model protein, urea-denatured/dithiothreitol-reduced lysozyme. Many factors influencing the refolding of lysozyme, such as urea concentration, beta-cyclodextrin concentration, molar ratio of detergent to protein, mobile phase flow rate, and type of detergent, were investigated, respectively, to optimize the conditions for lysozyme refolding by AMC-IEC. Compared with normal IEC refolding method, the activity recoveries of lysozyme obtained by AMC-IEC were much higher in the investigated range of initial protein concentrations. Moreover, the activity recoveries obtained by using this newly developed refolding method were still quite high for denatured/reduced lysozyme at high initial concentrations. When the initial protein concentration was 200 mg mL(-1), the activity recovery was over 60%. In addition, the lifetime of the chromatographic column during AMC-IEC was much longer than that during protein refolding by normal IEC. Therefore, AMC-IEC is a high efficient and low cost protein refolding method.

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“…This allowed simultaneous structural rearrangement and elution of denatured lysozyme during its migration along the column, thereby resulting in improved refolding yield and mass recovery (Li et al, 2002). Refolding recovery at high protein concentration was improved by another dualgradient IEC process wherein denatured hen egg-white lysozyme bound to the IEC matrix was gently eluted with refolding buffer employing decreasing urea (from 6 to 1 M) and increasing pH (from 6.2 to 10) gradients (Gu et al, 2002). This is in line with the finding that pH of the refolding buffer exhibited critical effects on the formation of disulfide bonds (Misawa et al, 1994).…”

Section: Column Refolding Using Ion Exchange Chromatography (Iec)mentioning

confidence: 99%

“…A new SEC refolding concept based on a dual-gradient of denaturant and pH was introduced (Gu et al, 2002) where dual gradient allowed solubilised-denatured recombinant scFv fragment to gradually experience lower denaturant concentrations and higher pH along the SEC column, thereby promoting refolding into native conformation. One major advantage of SEC for refolding in this particular mode is that the rate of removal of denaturant and the change in pH can be carefully controlled by adjusting the gradient slope and the elution flow rate.…”

Section: Column Refolding Using Size Exclusion Chromatography (Sec)mentioning

confidence: 99%

Recent advances in biomolecular process intensification

Choe

Nian

Lai

2006

Chemical Engineering Science

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Chromatographic Methods for the Isolation of, and Refolding of Proteins from, Escherichia coli Inclusion Bodies

Cited by 71 publications

References 13 publications

Cloning and Expression of Human Stem Cell Factor Fused with Thrombopoietin Mimetic Peptide in Escherichia coli

Cloning and Expression of Human Stem Cell Factor Fused with Thrombopoietin Mimetic Peptide in Escherichia coli

Refolding of denatured/reduced lysozyme at high concentrations by artificial molecular chaperone‐ion exchange chromatography

Recent advances in biomolecular process intensification

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