Continuous refolding of lysozyme with fed-batch addition of denatured protein solution

Katoh, Shigeo; Katoh, Yoshihiro

doi:10.1016/s0032-9592(00)00145-x

Cited by 58 publications

(24 citation statements)

References 7 publications

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“…As unfolded protein never aggregates with folded protein, pulsatile refolding reduced the extent of protein aggregation and resulted in improved refolding of the solubilized proteins. 34,35 Folding follows first-order kinetics whereas aggregation follows multiple-order kinetics. Therefore a decline in the concentration of folding intermediates was of paramount importance for optimal renaturation of solubilized protein.…”

Section: 33mentioning

confidence: 99%

Solubilization at high pH results in improved recovery of proteins from inclusion bodies of E. coli

Singh

Upadhyay

Panda

2008

J of Chemical Tech & Biotech

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BACKGROUND: Solubilization of inclusion bodies using high concentration of chaotropes followed by suboptimal refolding results in lower recovery of bioactive proteins from E. coli. Growing evidence indicates that aggregation of expressed proteins into inclusion bodies is molecular specific in nature and proteins in inclusion bodies have native-like secondary structure. Protecting these pre-existing native-like secondary structures of proteins using mild solubilization conditions is one of the key steps in improved recovery of bioactive protein from inclusion bodies of E. coli.

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Section: 33mentioning

confidence: 99%

Solubilization at high pH results in improved recovery of proteins from inclusion bodies of E. coli

Singh

Upadhyay

Panda

2008

J of Chemical Tech & Biotech

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“…To avoid protein precipitation during refolding at high protein concentrations, the solubilized, denatured protein can be added in pulses or continuously to the refolding buffer, thereby avoiding the accumulation of aggregation-prone folding intermediates (Buchner et al, 1992;Fischer et al, 1992;Katoh and Katoh, 2000;Terashima et al, 1996). Refolding attempts were started with denatured and reduced rhBMP-2 obtained directly from washed inclusion bodies as described in Materials and Methods.…”

Section: Pulse Refoldingmentioning

confidence: 99%

Optimized procedure for renaturation of recombinant human bone morphogenetic protein‐2 at high protein concentration

Vallejo

Rinas

2004

Biotech & Bioengineering

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The human gene encoding the mature form of bone morphogenetic protein-2 (hBMP-2), a dimeric disulfide-bonded protein of the cystine knot growth factor family, was expressed in recombinant Escherichia coli using a temperature-inducible expression system. The recombinant protein was produced in the form of cytoplasmic inclusion bodies and the effect of different variables on the renaturation of rhBMP-2 was investigated. In particular, variables such as pH, redox conditions, protein concentration, temperature, the presence of different types of aggregation suppressors, and host cell contaminants were studied with respect to their effect on aggregation during refolding and on the final renaturation yield of rhBMP-2. It is shown that the renaturation yield is particularly sensitive to pH, temperature, protein concentration, and the presence of aggregation suppressors. In contrast, little effect of the redox conditions and the ionic strength on the renaturation yield was observed, as equal yields were obtained in a broad range of reduced to oxidized glutathione ratios and concentrations of NaCl, respectively. The aggregation suppressor 2-(cyclohexylamino)ethanesulfonic acid (CHES) proved to be superior with respect to the final renaturation yield, although, in comparison to the more common arginine, it was less efficient in preventing aggregation of rhBMP-2 during refolding. Detergent washing of inclusion bodies was sufficient, as further purification of rhBMP-2 prior to refolding was without effect on the final renaturation yield. An increase in the concentration of renatured rhBMP-2 was achieved by a pulsed refolding procedure by which up to a total amount of 2.1 mg mL(-1) rhBMP-2 could be transferred in seven pulses into the renaturation buffer with an overall refolding yield of 38%, corresponding to 0.8 mg mL(-1) renatured dimeric rhBMP-2. Furthermore, a simplified purification procedure is presented that also includes freeze-drying for long-term storage of biologically active rhBMP-2. Finally, it is shown that the appearance of rhBMP-2 variants could be avoided by using a host strain overexpressing rare codon tRNAs.

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“…Various other groups have found that slower addition of the denatured protein to refolding buffer gives improved yields (Katoh et al, 1999;Katoh and Katoh, 2000). Under such slow injection rates, it is likely that the influence of macroscopic concentration gradients will be minimized even under poor mixing conditions.…”

Section: Introductionmentioning

confidence: 99%

A critical assessment of the impact of mixing on dilution refolding

Mannall

Titchener‐Hooker

Chase

et al. 2005

Biotech & Bioengineering

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Refolding often presents a bottleneck in the generation of recombinant protein expressed as inclusion bodies. Few studies have looked at the effect of physical factors on the yield from refolding steps. Refold reactors typically operate in fed-batch mode with a slow injection rate. This paper characterizes mixing in a novel reactor, and seeks to relate the conditions of mixing to native lysozyme yields after refolding. A novel twin-impeller system incorporating a mini-paddle impeller located in the vicinity of the injection point was used to increase the local levels of energy dissipation experienced by the injected material, and to improve refolding yields. Mixing only affected yields during and immediately after denatured protein addition. Analysis of lysozyme refolding yield, under a variety of conditions, revealed that dispersive mixing affected the yield. The beneficial effect of the mini-paddle impeller in providing a source of localized energy dissipation was limited to conditions where the bulk impeller intensity was low. The effects appeared to become more significant when injection times were longer, because of increased exposure of the injected material to the energy dissipation of the mini-impeller. The results suggest that for fed-batch protein refolding systems, where mixing has been shown to be a critical factor, the local energy dissipation experienced in the vicinity of the injection point is critical to the refolding yields.

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Continuous refolding of lysozyme with fed-batch addition of denatured protein solution

Cited by 58 publications

References 7 publications

Solubilization at high pH results in improved recovery of proteins from inclusion bodies of E. coli

Solubilization at high pH results in improved recovery of proteins from inclusion bodies of E. coli

Optimized procedure for renaturation of recombinant human bone morphogenetic protein‐2 at high protein concentration

A critical assessment of the impact of mixing on dilution refolding

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