Urea gradient size-exclusion chromatography enhanced the yield of lysozyme refolding

Gu, Zhenyu; Su, Zhiguo; Janson, Jan‐Christer

doi:10.1016/s0021-9673(01)00766-x

Cited by 122 publications

(86 citation statements)

References 17 publications

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“…A linear decrease of urea concentration during diafiltration was very effective on obtaining increased refolding recovery at relatively high protein concentration [13]. Gu et al [14] have demonstrated that gradient decrease of urea concentration during size-exclusion chromatography gives high activity recovery at relatively high protein concentration.…”

Section: Introductionmentioning

confidence: 99%

Refolding human lysozyme produced as an inclusion body by urea concentration and pH gradient ion exchange chromatography

2002

Chromatographia

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SummaryA new dual-gradient ion exchange chromatographic method was developed to improve the refolding yield of human lysozyme produced in Escherichia coil as an inclusion body. The dissolved and stretched polypeptide chain in a concentrated non-ionic denaturant was adsorbed onto an ion exchange column and induced to refold by gradually decreasing the denatu rant concentration and increasing pH in the flowing buffer. The dual gradients of denaturant concentration and pH provided a gradual change of the solution environment along the chromatographic column for the protein to refold, resulting in enhanced activityyield and purity.A post-separation was also studied using size-exclusion chromatography to remove protein aggregates and mis-folded proteins after the refolding step.

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Section: Introductionmentioning

confidence: 99%

Refolding human lysozyme produced as an inclusion body by urea concentration and pH gradient ion exchange chromatography

2002

Chromatographia

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“…Different refolding processes were investigated such as twogradient size-exclusion chromatography process, urea-only gradient size-exclusion chromatography refolding process (Gu and Su, 2001), non-gradient size-exclusion chromatography refolding system (SEPROS) (Batas and Chaudhuri, 1995) and dilution refolding process. All dilution factors (a dilution factor of 55) were the same in different refolding processes.…”

Section: Refolding Of Solubilized Rhnta Proteinmentioning

confidence: 99%

“…Thus, as a low-molecular-weight additive, arginine, was usually used in the dilution refolding process, especially in the refolding of protein containing many cysteine residues to suppress aggregation. Size-exclusion chromatography refolding systems have been successfully developed to inhibit aggregation and increase refolding yield in recent years (Batas and Chaudhuri, 1995;Gu and Su, 2001;Dong et al, 2002). However, the refolding technique coupling arginine with size-exclusion gel filtration process has not previously been reported.…”

Section: Refolding Of Solubilized Rhnta Proteinmentioning

confidence: 99%

“…Refolding at such low protein concentration requires long refolding time and large volumes of refolding buffer. Instead of dilution refolding, some chromatographic refolding systems have been successfully developed to inhibit aggregation and increase refolding yield in recent years (Batas and Chaudhuri, 1995;Gu and Su, 2001;Dong et al, 2002). Size-exclusion chromatography, as one of the wellknown chromatography techniques, can be used to separate proteins of different molecular mass, realizing refolding and simultaneous purification.…”

Section: Introductionmentioning

confidence: 99%

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Refolding and purification of rhNTA protein by chromatography

Fan¹,

Xu²,

Lü³

et al. 2008

Biomedical Chromatography

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RhNTA protein is a new thrombolytic agent which has potential medicinal and commercial value. Protein refolding is a bottleneck for large-scale production of valuable proteins expressed as inclusion bodies in Escherichia coli. The denatured rhNTA protein was refolded by an improved size-exclusion chromatography refolding process achieved by combining an increasing arginine gradient and a decreasing urea gradient (two gradients) with a size-exclusion chromatography refolding system. The refolding of denatured rhNTA protein showed that this method could significantly increase the activity recovery of protein at high protein concentration. The activity recovery of 37% was obtained from the initial rhNTA protein concentration up to 20 mg/mL. After refolding by two-gradient size-exclusion chromatography refolding processes, the refolded rhNTA was purified by ion-exchange and affinity chromatography. The purified rhNTA protein showed one band in SDS-PAGE and the specific activity of purified rhNTA protein was 110,000 U/mg.

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“…The use of size-exclusion chromatography for refolding was further improved by Gu et al, who used urea gradients to guarantee the gradual transfer of the unfolded protein to refolding conditions [159]. In a very thorough analysis, chromatographic refolding methods were compared to more traditional refolding techniques using E. coli inclusion bodies of recombinant single-chain antibody fragments [160].…”

Section: Folding By Gel Filtrationmentioning

confidence: 99%

Production of Recombinant Proteins for Therapy, Diagnostics, and Industrial Research by in Vitro Folding

Lange¹,

Rudolph²

2005

Protein Folding Handbook

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Urea gradient size-exclusion chromatography enhanced the yield of lysozyme refolding

Cited by 122 publications

References 17 publications

Refolding human lysozyme produced as an inclusion body by urea concentration and pH gradient ion exchange chromatography

Refolding human lysozyme produced as an inclusion body by urea concentration and pH gradient ion exchange chromatography

Refolding and purification of rhNTA protein by chromatography

Production of Recombinant Proteins for Therapy, Diagnostics, and Industrial Research by in Vitro Folding

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