Rhodanese folding is controlled by the partitioning of its folding intermediates

Gorovits, Boris; McGee, William A.; Horowitz, Paul M.

doi:10.1016/s0167-4838(97)00158-1

Cited by 24 publications

(45 citation statements)

References 18 publications

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“…GroEL can also bind and reactivate some folding intermediates, which otherwise would not fold to give active protein. A model for the spontaneous folding of rhodanese has been proposed previously (1,2). We use and extend this model to interpret the present studies.…”

mentioning

confidence: 54%

“…Model for Rhodanese Folding-A model has been proposed for the spontaneous folding of rhodanese (1,2). In the present work, we use this model to interpret our data, and for convenience, it is presented in Scheme 1.…”

Section: Resultsmentioning

confidence: 99%

“…Even under these conditions, some inactive, monomeric species are formed, so that 100% reactivation is never achieved. Urea-induced denaturation of rhodanese involves steps that include domain separation followed by complete denaturation of the protein (1,14,15). The domain-dissociated intermediate contains a high degree of secondary structure, and it displays extensive hydrophobic exposure (14,16,17).…”

mentioning

confidence: 99%

“…The domain-dissociated intermediate contains a high degree of secondary structure, and it displays extensive hydrophobic exposure (14,16,17). Consequently, it can form large aggregates that can be monitored by light scattering (1,17). Aggregation of rhodanese can be slowed by limiting the collision among interactive intermediates by lowering the protein concentration (13) including detergent micelles in the reactivation solutions (18), lowering the temperature (19), or by binding partially folded intermediates to molecular chaperones such as GroEL (20 -24).…”

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confidence: 99%

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The Aggregation State of Rhodanese during Folding Influences the Ability of GroEL to Assist Reactivation

Bhattacharyya

Horowitz

2001

Journal of Biological Chemistry

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The in vitro folding of rhodanese involves a competition between formation of properly folded enzyme and offpathway inactive species. Co-solvents like glycerol or low temperature, e.g. refolding at 10°C, successfully retard the off-pathway formation of large inactive aggregates, but the process does not yield 100% active enzyme. These data suggest that mis-folded species are formed from early folding intermediates. GroEL can capture early folding intermediates, and it loses the ability to capture and reactivate rhodanese if the enzyme is allowed first to spontaneously fold for longer times before it is presented to GroEL, a process that leads to the formation of unproductive intermediates. In addition, GroEL cannot reverse large aggregates once they are formed, but it could capture some folding intermediates and activate them, even though they are not capable of forming active enzyme if left to spontaneous refolding. The interaction between GroEL and rhodanese substantially but not completely inhibits intra-protein inactivation, which is responsible for incomplete activation during unassisted refolding. Thus, GroEL not only decreases aggregation, but it gives the highest reactivation of any method of assistance. The results are interpreted using a previously suggested model based on studies of the spontaneous folding of rhodanese (Gorovits, B. M., McGee, W.

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confidence: 54%

Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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The Aggregation State of Rhodanese during Folding Influences the Ability of GroEL to Assist Reactivation

Bhattacharyya

Horowitz

2001

Journal of Biological Chemistry

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“…Glycerol was also known to stabilize proteins against chemical and thermal denaturation (Timasheff 2002). Additionally, glycerol has been used to increase the refolding yield of proteins such as citrate synthase, thiosulfate sulfurtransferase, pancreatic elastase, and malate dehydrogenases in the in-vitro studies (Gorovits et al 1998;Jaspard 2000;Mishra et al 2005;Tieman et al 2001). Glycerol also prevents human binterferon aggregation during synthesis but does not dissociate pre-formed aggregates in CHO cells (Rodriguez et al 2005).…”

Section: Effects Of Glycerolmentioning

confidence: 99%

Enhanced recombinant M-CSF production in CHO cells by glycerol addition: model and validation

Liu

Chen

2007

Cytotechnology

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Addition of stimulatory chemical such as glycerol was found to increase recombinant protein production in Chinese hamster ovary (CHO) cells. However, glycerol influenced cell mitosis and reduced cell growth rate. We developed a controlled proliferation strategy to utilize the stimulation of glycerol on recombinant protein production and mitigate the problem of growth inhibition. The approach is to apply a two-stage process, where cells are cultured without glycerol for a period of time in order to obtain enough cell density and then glycerol is added to achieve high specific productivity. In addition, a model for predicting the profiles of cell proliferation and recombinant protein production was developed and validated. A two-stage process, addition of 1% glycerol after 1 day of growth, could increase the final production of macrophage-colony stimulating factor (M-CSF) by 38% compared with the value obtained without addition of glycerol.

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Synergistic coordination of polyethylene glycol with ClpB/DnaKJE bichaperone for refolding of heat‐denatured malate dehydrogenase

Nian

Kim

Tan

et al. 2009

Biotechnology Progress

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The use of polyethylene glycol (PEG) as a refolding additive to a refolding cocktail comprising the molecular bichaperone ClpB and DnaKJE significantly enhances chaperone-mediated refolding of heat-denatured malate dehydrogenase (MDH). The critical factor to affect the refolding yield is the time point of introducing PEG to the refolding cocktail. The refolding efficiency reached approximately 90% only when PEG was added at the beginning of refolding reaction. The synergistic coordination of an inexpensive refolding additive PEG with the ClpB/DnaKJE bichaperone system may provide an economical route to further enhance the efficacy of ClpB/DnaKJE refolding cocktail approach, facilitating its implementation in large-scale refolding processes.

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Rhodanese folding is controlled by the partitioning of its folding intermediates

Cited by 24 publications

References 18 publications

The Aggregation State of Rhodanese during Folding Influences the Ability of GroEL to Assist Reactivation

The Aggregation State of Rhodanese during Folding Influences the Ability of GroEL to Assist Reactivation

Enhanced recombinant M-CSF production in CHO cells by glycerol addition: model and validation

Synergistic coordination of polyethylene glycol with ClpB/DnaKJE bichaperone for refolding of heat‐denatured malate dehydrogenase

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