Ionic Liquids as Protein Crystallization Additives

Tarver, Crissy L; Yuan, Qunying; Pusey, Marc L.

doi:10.3390/cryst11101166

Cited by 9 publications

(7 citation statements)

References 44 publications

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“…It was also reported that for a long storage period of 72 h at −80 °C, hMBCOMT still presents an increment of 27% on its original activity recovery. Moreover, if 4 °C was considered to preserve the protein, the addition of low concentrations of [Ch][DHP] maintained the enzyme’s biological activity for 48 h. Therefore, this stabilization formulation can favor later crystallization and bio-interaction trials, while disfavoring the precipitation of the additives present at higher concentrations [ 48 ]. Herein, and for the soluble isoform hSCOMTVal108-6His, both 4 and −80 °C temperatures were evaluated after a period of 72 h of storage ( Table 1 ) using protein samples solubilized in both Buffer A and Buffer B with and without glycerol.…”

Section: Resultsmentioning

confidence: 99%

Thermofluor-Based Optimization Strategy for the Stabilization of Recombinant Human Soluble Catechol-O-Methyltransferase

Gonçalves

Pedro

Oliveira

et al. 2022

IJMS

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Catechol-O-methyltransferase (COMT) has been involved in a number of medical conditions including catechol-estrogen-induced cancers and a great range of cardiovascular and neurodegenerative diseases such as Parkinson’s disease. Currently, Parkinson’s disease treatment relies on a triple prophylaxis, involving dopamine replacement by levodopa, the use of aromatic L-amino acid decarboxylase inhibitors, and the use of COMT inhibitors. Typically, COMT is highly thermolabile, and its soluble isoform (SCOMT) loses biological activity within a short time span preventing further structural and functional trials. Herein, we characterized the thermal stability profile of lysate cells from Komagataella pastoris containing human recombinant SCOMT (hSCOMT) and enzyme-purified fractions (by Immobilized Metal Affinity Chromatography—IMAC) upon interaction with several buffers and additives by Thermal Shift Assay (TSA) and a biological activity assessment. Based on the obtained results, potential conditions able to increase the thermal stability of hSCOMT have been found through the analysis of melting temperature (Tm) variations. Moreover, the use of the ionic liquid 1-butyl-3-methylimidazolium chloride [C4mim]Cl (along with cysteine, trehalose, and glycerol) ensures complete protein solubilization as well as an increment in the protein Tm of approximately 10 °C. Thus, the developed formulation enhances hSCOMT stability with an increment in the percentage of activity recovery of 200% and 70% when the protein was stored at 4 °C and −80 °C, respectively, for 12 h. The formation of metanephrine over time confirmed that the enzyme showed twice the productivity in the presence of the additive. These outstanding achievements might pave the way for the development of future hSCOMT structural and biophysical studies, which are fundamental for the design of novel therapeutic molecules.

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Section: Resultsmentioning

confidence: 99%

Thermofluor-Based Optimization Strategy for the Stabilization of Recombinant Human Soluble Catechol-O-Methyltransferase

Gonçalves

Pedro

Oliveira

et al. 2022

IJMS

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“…Ionic liquids, which are composed of cations and anions, are liquid molten salts at room temperature. Owing to their advantages of low volatility, high viscosity and thermal stability, and excellent solvation ability and ionic strength, ionic liquids have been used as solvents or additives during the crystallization process . It is worth noting that several ionic liquids can be recurrently used for the polymorph control of API.…”

Section: Novel Techniques For Preparation and Regulation Of Drug Poly...mentioning

confidence: 99%

An Overview on Polymorph Preparation Methods of Active Pharmaceutical Ingredients

Zhou,

Lv,

Liu

et al. 2023

Crystal Growth & Design

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Polymorphism, which plays an important role in the pharmaceutical industry, is quite common for active pharmaceutical ingredients (API). Owing to the structural differences between polymorphs, they often possess remarkably different physical and chemical properties, thus leading to diversity in curative effects and shelf life. How to precisely control the polymorph of API has attracted the attention of researchers around the world. In this paper, the regulation methods of drug polymorphs are comprehensively reviewed, to give a better view for the future research. The traditional method, including the control of the nucleation and growth process, is systematically introduced and analyzed from the aspects of the action mechanism and their application in recent years. In addition, novel techniques, including templates, microfluidics technology, ultrasound, laser, ionic liquids, and supramolecular gels, are expounded and analyzed in detail. Considerable studies indicate that polymorph control of API is a promising research field. Finally, the existing problems are discussed and the future prospects are given.

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“…The first was the choline-based IL, which was extensively studied to reveal whether it promotes or inhibits protein aggregation and protein stabilization, with its outcome being IL concentration dependent [28,46,47]. The second was the imidazolium-based IL, which was employed as a co-solvent or an additive for protein crystallization as shown by the group in the stabilization of periplasmic molybdenum aldehyde oxidoreductase (PaoD) protein in the presence of [C4mim]Cl, with its effect being alkyl chain-length dependent [32,48]. Moreover, as shown in Table 1, beyond the study of the two different cation classes, we also analyzed a wide range of IL concentrations, between 5 to 500 mM and with different anion paring, since both cationic and anionic parts of IL can play an important role in protein stabilization [35].…”

Section: Preliminary Studies For Hmbcomt Stabilizationmentioning

confidence: 99%

Advances in Membrane-Bound Catechol-O-Methyltransferase Stability Achieved Using a New Ionic Liquid-Based Storage Formulation

Gonçalves

Sousa

Pedro

et al. 2022

IJMS

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Membrane-bound catechol-O-methyltransferase (MBCOMT), present in the brain and involved in the main pathway of the catechol neurotransmitter deactivation, is linked to several types of human dementia, which are relevant pharmacological targets for new potent and nontoxic inhibitors that have been developed, particularly for Parkinson’s disease treatment. However, the inexistence of an MBCOMT 3D-structure presents a blockage in new drugs’ design and clinical studies due to its instability. The enzyme has a clear tendency to lose its biological activity in a short period of time. To avoid the enzyme sequestering into a non-native state during the downstream processing, a multi-component buffer plays a major role, with the addition of additives such as cysteine, glycerol, and trehalose showing promising results towards minimizing hMBCOMT damage and enhancing its stability. In addition, ionic liquids, due to their virtually unlimited choices for cation/anion paring, are potential protein stabilizers for the process and storage buffers. Screening experiments were designed to evaluate the effect of distinct cation/anion ILs interaction in hMBCOMT enzymatic activity. The ionic liquids: choline glutamate [Ch][Glu], choline dihydrogen phosphate ([Ch][DHP]), choline chloride ([Ch]Cl), 1- dodecyl-3-methylimidazolium chloride ([C12mim]Cl), and 1-butyl-3-methylimidazolium chloride ([C4mim]Cl) were supplemented to hMBCOMT lysates in a concentration from 5 to 500 mM. A major potential stabilizing effect was obtained using [Ch][DHP] (10 and 50 mM). From the DoE 146% of hMBCOMT activity recovery was obtained with [Ch][DHP] optimal conditions (7.5 mM) at −80 °C during 32.4 h. These results are of crucial importance for further drug development once the enzyme can be stabilized for longer periods of time.

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Ionic Liquids as Protein Crystallization Additives

Cited by 9 publications

References 44 publications

Thermofluor-Based Optimization Strategy for the Stabilization of Recombinant Human Soluble Catechol-O-Methyltransferase

Thermofluor-Based Optimization Strategy for the Stabilization of Recombinant Human Soluble Catechol-O-Methyltransferase

An Overview on Polymorph Preparation Methods of Active Pharmaceutical Ingredients

Advances in Membrane-Bound Catechol-O-Methyltransferase Stability Achieved Using a New Ionic Liquid-Based Storage Formulation

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