Effects of ionic strength on inclusion body refolding at high concentration

Gabrielczyk, Jakub; Kluitmann, Jonas; Dammeyer, Thorben; Jördening, Hans‐Joachim

doi:10.1016/j.pep.2016.10.004

Cited by 12 publications

(7 citation statements)

References 37 publications

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“…It is known that the ionic strength determines the protein folding in solution (Gabrielczyk et al, 2017) and influences on the ligand-protein binding affinity (Papaneophytou et al, 2014). As mentioned above, there are evidences of benefits attributed to the inhalation of a hypertonic saline solution (HTS) in the treatment of patients with lung diseases.…”

Section: Molecular Dynamic Simulationsmentioning

confidence: 99%

Anin silicoanalysis of Ibuprofen enantiomers in high concentrations of sodium chloride with SARS-CoV-2 main protease

Clemente

Freiberger

Ravetti

et al. 2021

Journal of Biomolecular Structure and Dynamics

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2020 will be remembered worldwide for the outbreak of Coronavirus disease (COVID-19), which quickly spread until it was declared as a global pandemic. The main protease (Mpro) of SARS-CoV-2, a key enzyme in coronavirus, represents an attractive pharmacological target for inhibition of SARS-CoV-2 replication. Here, we evaluated whether the anti-inflammatory drug Ibuprofen, may act as a potential SARS-CoV-2 Mpro inhibitor, using an in silico study. From molecular dynamics (MD) simulations, we also evaluated the influence of ionic strength on the affinity and stability of the Ibuprofen-Mpro complexes. The docking analysis shows that R(À)Ibuprofen and S(þ)Ibuprofen isomers can interact with multiple key residues of the main protease, through hydrophobic interactions and hydrogen bonds, with favourable binding energies (À6.2 and À5.7 kcal/mol, respectively). MM-GBSA and MM-PBSA calculations confirm the affinity of these complexes, in terms of binding energies. It also demonstrates that the ionic strength modifies significantly their binding affinities. Different structural parameters calculated from the MD simulations (120 ns) reveal that these complexes are conformational stable in the different conditions analysed. In this context, the results suggest that the condition 2 (0.25 NaCl) bind more tightly the Ibuprofen to Mpro than the others conditions. From the frustration analysis, we could characterize two important regions (Cys44-Pro52 and Linker loop) of this protein involved in the interaction with Ibuprofen. In conclusion, our findings allow us to propose that racemic mixtures of the Ibuprofen enantiomers might be a potential treatment option against SARS-CoV-2 Mpro. However, further research is necessary to determinate their possible medicinal use.

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Section: Molecular Dynamic Simulationsmentioning

confidence: 99%

Anin silicoanalysis of Ibuprofen enantiomers in high concentrations of sodium chloride with SARS-CoV-2 main protease

Clemente

Freiberger

Ravetti

et al. 2021

Journal of Biomolecular Structure and Dynamics

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“…The highest yield of the dimers was observed in the refolding buffers that contained an ionic detergent such as sarkosyl or CPC (Figure A, xii). Gabrielczyk et al refolded the fructosyltransferase enzyme and found renaturation of the unfolded protein depending on the ionic strength and the cosolvent charge density . Mizukami et al reported that increasing the ionic strength could accelerate refolding and slow down unfolding of SAMP1 proteins .…”

Section: Discussionmentioning

confidence: 99%

Effects of Chemical Additives in Refolding Buffer on Recombinant Human BMP-2 Dimerization and the Bioactivity on SaOS-2 Osteoblasts

et al. 2023

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Bone morphogenetic protein-2 (BMP-2) is a promising osteogenic agent in tissue engineering. BMP-2 is usually expressed in Escherichia coli owing to the high yield and low cost, but the protein is expressed as inclusion bodies. Thus, the bottleneck for BMP-2 production in E. coli is the refolding process. Here, we explored the effects of the refolding buffer composition on BMP-2 refolding. The BMP-2 inclusion body was solubilized in urea and subjected to refolding by the dilution method. Various additives were investigated to improve the BMP-2 refolding yield. Nonreducing SDS-PAGE showed that BMP-2 dimers, the presumably biologically active form, were detected at approximately 25 kDa. The highest yield of the BMP-2 dimers was observed in the refolding buffer that contained ionic detergents (sarkosyl and cetylpyridinium chloride) followed by zwitterionic and nonionic detergents (NDSB-195, NP-40, and Tween 80). In addition, sugars (glucose, sorbitol, and sucrose) in combination with anionic detergents (sodium dodecyl sulfate and sarkosyl) reduced BMP-2 oligomers and increased the BMP-2 dimer yield. Subsequently, the refolded BMP-2s were tested for their bioactivity using the alkaline phosphatase assay in osteogenic cells (SaOS-2), as well as the luciferase reporter assay and the calcium assays. The refolded BMP-2 showed the activities in the calcium deposition assay and the luciferase reporter assay but not in the alkaline phosphatase activity assay or the intracellular calcium assay even though the dimers were clearly detected. Therefore, the detection of the disulfide-linked dimeric BMP-2 in nonreducing SDS-PAGE is an inadequate proxy for the bioactivity of BMP-2.

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“…We favored a fast protocol, in which relevant conditions for protein refolding were assayed. In this respect, the most studied variables in the culture medium are NaCl concentration, since it can stabilize proteins by either hydration or exclusion of water 31 ; the presence of arginine, which aids the refolding process, although its exact mechanism is still unclear 32 ; and glycerol, which enhances hydrophobic interactions by ordering the solvent around the protein 32 . The use of the redox pair GSH/GSSG was considered as the scFv structures tend to have disulfide bonds 33 .…”

Section: Discussionmentioning

confidence: 99%

Optimization of culture conditions for the expression of three different insoluble proteins in Escherichia coli

Gutiérrez-González

Farías

Tello

et al. 2019

Sci Rep

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Recombinant protein expression for structural and therapeutic applications requires the use of systems with high expression yields. Escherichia coli is considered the workhorse for this purpose, given its fast growth rate and feasible manipulation. However, bacterial inclusion body formation remains a challenge for further protein purification. We analyzed and optimized the expression conditions for three different proteins: an anti-MICA scFv, MICA, and p19 subunit of IL-23. We used a response surface methodology based on a three-level Box-Behnken design, which included three factors: post-induction temperature, post-induction time and IPTG concentration. Comparing this information with soluble protein data in a principal component analysis revealed that insoluble and soluble proteins have different optimal conditions for post-induction temperature, post-induction time, IPTG concentration and in amino acid sequence features. Finally, we optimized the refolding conditions of the least expressed protein, anti-MICA scFv, using a fast dilution protocol with different additives, obtaining soluble and active scFv for binding assays. These results allowed us to obtain higher yields of proteins expressed in inclusion bodies. Further studies using the system proposed in this study may lead to the identification of optimal environmental factors for a given protein sequence, favoring the acceleration of bioprocess development and structural studies.

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Effects of ionic strength on inclusion body refolding at high concentration

Cited by 12 publications

References 37 publications

Anin silicoanalysis of Ibuprofen enantiomers in high concentrations of sodium chloride with SARS-CoV-2 main protease

Anin silicoanalysis of Ibuprofen enantiomers in high concentrations of sodium chloride with SARS-CoV-2 main protease

Effects of Chemical Additives in Refolding Buffer on Recombinant Human BMP-2 Dimerization and the Bioactivity on SaOS-2 Osteoblasts

Optimization of culture conditions for the expression of three different insoluble proteins in Escherichia coli

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