Expression, refolding and purification of a human interleukin-17A variant

Wu, Bingyuan; Nemeth, Jennifer F.; Janecki, Dariusz; Jones, Brian H.; Obmolova, Galina; Malia, T.; Baker, Audrey; Bethea, Deidra; Elloso, M. Merle; Naso, Michael F.; Taudte, Susann

doi:10.1016/j.cyto.2010.07.002

Cited by 13 publications

(17 citation statements)

References 15 publications

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“…As IL-17A has been previously refolded in buffer with redox couples, the impact of them on the refolding was further explored 34 . By adding 5 mM cysteine and 0.5 mM cystine to the refolding buffer described above, we were surprised to see a clear transition curve when the protein was incubated in refolding buffer for only five min (Fig.…”

Section: Resultsmentioning

confidence: 99%

A Systematic Protein Refolding Screen Method using the DGR Approach Reveals that Time and Secondary TSA are Essential Variables

Wang

Oosterwijk

Ali

et al. 2017

Sci Rep

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Refolding of proteins derived from inclusion bodies is very promising as it can provide a reliable source of target proteins of high purity. However, inclusion body-based protein production is often limited by the lack of techniques for the detection of correctly refolded protein. Thus, the selection of the refolding conditions is mostly achieved using trial and error approaches and is thus a time-consuming process. In this study, we use the latest developments in the differential scanning fluorimetry guided refolding approach as an analytical method to detect correctly refolded protein. We describe a systematic buffer screen that contains a 96-well primary pH-refolding screen in conjunction with a secondary additive screen. Our research demonstrates that this approach could be applied for determining refolding conditions for several proteins. In addition, it revealed which “helper” molecules, such as arginine and additives are essential. Four different proteins: HA-RBD, MDM2, IL-17A and PD-L1 were used to validate our refolding approach. Our systematic protocol evaluates the impact of the “helper” molecules, the pH, buffer system and time on the protein refolding process in a high-throughput fashion. Finally, we demonstrate that refolding time and a secondary thermal shift assay buffer screen are critical factors for improving refolding efficiency.

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Section: Resultsmentioning

confidence: 99%

A Systematic Protein Refolding Screen Method using the DGR Approach Reveals that Time and Secondary TSA are Essential Variables

Wang

Oosterwijk

Ali

et al. 2017

Sci Rep

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“…Incorporation of osmolytes such as amino acids, sugars, and low concentrations of chaotropes in the renaturation buffer results in high refolding yield of recombinant proteins. Arginine has been one of the most commonly used folding aids for recovery of soluble proteins from inclusion bodies (35,40). In all these cases, overexpression of protein involves formation of inclusion bodies from which the soluble protein can be recovered using multiple steps.…”

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confidence: 99%

Effect of Chemical Chaperones in Improving the Solubility of Recombinant Proteins in Escherichia coli

Prasad

Khadatare

Roy

2011

Appl Environ Microbiol

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The recovery of active proteins from inclusion bodies usually involves chaotrope-induced denaturation, followed by refolding of the unfolded protein. The efficiency of renaturation is low, leading to reduced yield of the final product. In this work, we report that recombinant proteins can be overexpressed in the soluble form in the host expression system by incorporating compatible solutes during protein expression. Green fluorescent protein (GFP), which was otherwise expressed as inclusion bodies, could be made to partition off into the soluble fraction when sorbitol and arginine, but not ethylene glycol, were present in the growth medium. Arginine and sorbitol increased the production of soluble protein, while ethylene glycol did not. Production of ATP increased in the presence of sorbitol and arginine, but not ethylene glycol. A control experiment with fructose addition indicated that protein solubilization was not due to a simple ATP increase. We have successfully reproduced these results with the N-terminal domain of HypF (HypF-N), a bacterial protein which forms inclusion bodies in Escherichia coli. Instead of forming inclusion bodies, HypF-N could be expressed as a soluble protein in the presence of sorbitol, arginine, and trehalose in the expression medium.Overexpression of proteins in heterologous systems often results in the formation of inclusion bodies. These electronrefractile particles are formed because of the failure of prokaryotic folding machinery to correctly fold the nascent polypeptide chain, increasing its local concentration in the cytosol. The most common strategy to recover active proteins from these particles is by denaturing them in the presence of chaotropes and refolding them to the native conformation by the gradual removal of the denaturant. This process results in high loss in protein yield since the renaturation efficiency of most proteins is not very high.

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“…[30] Furthermore, arginine could improve the recovery of soluble protein from inclusion bodies. [31] CTAB, a cationic surfactant, is an amphipathic molecule which could reduce the aggregation of protein and increase the protein stability during cell disruption. There are also some reports about the application of surfactants in protein refolding studies.…”

Section: Discussionmentioning

confidence: 99%

Improvement of Soluble Production of Reteplase in Escherichia Coli by Optimization of Chemical Chaperones in Lysis Buffer

et al. 2019

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Background:Reteplase is a nonglycosylated derivative of recombinant tissue plasminogen activator, a thrombolytic agent, which can be easily expressed in Escherichia coli. However, overexpression of reteplase in E. coli usually leads to accumulation of insoluble and inactive aggregates and inclusion bodies. In the present study, we aimed to optimize chemical additives of lysis buffer to avoid the initial aggregation and formation of inclusion bodies of reteplase at cell disruption step.Materials and Methods:After protein expression in E. coli BL21 (DE3), the bacterial cells were disrupted in different lysis buffers using microsmashing. Eleven chemical additives at two concentration levels were combined based on a Plackett–Burman design to prepare 12 different lysis buffers used at cell disruption stage. Then, three additives with the most positive effect on improvement of solubility of reteplase were chosen and used for the second screening based on Box–Behnken model.Results:The primary screening results showed that among 11 additives, arginine, K2PO4, and cetyltrimethylammonium bromide (CTAB) had the most positive effect on solubility of reteplase. Our final results based on 14 runs of Box–Behnken design showed that the optimum buffer additive condition is 0.005 mg/ml CTAB, 0.065 mg/ml arginine, and 0.026 mg/ml K2PO4. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis and Western blotting of soluble and total fraction of samples confirmed that these additives significantly improved soluble production of reteplase compared with control.Conclusion:Our study indicates that the application of chemical additives in cell lysis can improve the solubility of reteplase. Further studies are still required to understand the exact mechanism of chemical additives as a chemical chaperone during cell lysis.

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Expression, refolding and purification of a human interleukin-17A variant

Cited by 13 publications

References 15 publications

A Systematic Protein Refolding Screen Method using the DGR Approach Reveals that Time and Secondary TSA are Essential Variables

A Systematic Protein Refolding Screen Method using the DGR Approach Reveals that Time and Secondary TSA are Essential Variables

Effect of Chemical Chaperones in Improving the Solubility of Recombinant Proteins in Escherichia coli

Improvement of Soluble Production of Reteplase in Escherichia Coli by Optimization of Chemical Chaperones in Lysis Buffer

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