Protein Refolding by N-Alkylpyridinium and N-Alkyl-N-methylpyrrolidinium Ionic Liquids

Abstract

An important property of ionic liquids consisting of cations and anions is that the chemical structures can be easily tuned. To expand the repertoire of effective ionic liquid-based refolding additives, we focused on this tunable property and investigated the effects of new candidates such as N-alkylpyridinium chlorides and N-alkyl-N-methylpyrrolidinium chlorides on protein refolding. Denatured lysozyme (30 mg/mL) was used as a model protein and refolded by 30-fold dilution with various refolding buffers containing different ionic liquids consisting of a systematic variety of alkyl chains. Compared with the refolding yield without additives (lower than 10%), less hydrophobic ionic liquids such as N-ethyl, N-butyl and N-hexylpyridinium chlorides, and N-butyl-N-methylpyrrolidinium chloride were effective in enhancing the refolding yields (46-69%), because they primarily suppressed aggregation because of their chaotropic properties. N-alkylpyridinium cations were more hydrophobic than N-alkyl-N-methylpyrrolidinium cations according to the calculated log P values and prevented aggregation at lower concentrations because of their hydrophobicity. The results provide a range of new effective ionic liquid-based additives for higher protein refolding yields and the knowledge of the effect of chemical structures of additives on protein refolding.

Proteins in Ionic Liquids: Current Status of Experiments and Simulations

“…However, 1-butyl-3-methylimidazolium acetate, nitrate and hexafluorophosphate have extremely low values of −2.8, −2.9 and −2.4 indicating they are much more polar than ethanol, which has . The value increases with increasing cationic alkyl chain length as expected [33]. …”

“…Although hydrophobic solvents are suggested to be more favorable for enzymatic reactions [34], which was also reported by [35], protein stability may decrease with rising hydrophobicity [33, 36]. Russell and co-workers [37] could not correlate values with the enzyme activity and argued that the anion is responsible for the reactivity, which points to nucleophilicity or hydrogen bond basicity [32].…”

Release Properties of Hydrogels: Water Evaporation from Alginate Gel Beads

“…At constant scanning rate of 5 K/min, thermograms obtained on beads with different composition show an exponential growth with different exponent that reflects the "obstructive" character of the polymer network in the bead. 13 First of all, there is a strong dependence of water evaporation on HPMC concentration; indeed the increase of HPMC is responsible for a decrease of the slope and water evaporation is delayed. The effect of reduced permeability of the matrix is supported also by release data of lysozyme.…”

Alumina nanoparticle-assisted enzyme refolding: A versatile methodology for proteins renaturation

“…In water, colloidal or interfacial assemblies with surfactants or nanoparticles provide conditions to control protein stability and folding/unfolding rearrangements and renaturation 3 . Several methods based on interface interactions for renaturation of proteins are available, which in general can be divided into three main groups: (I) methods to remove the denaturants from the protein solution 4–6 ; (II) methods involving the control of the physical conditions used during the refolding process 7, 8 ; and (III) methods in which refolding agents are added during the renaturing processes 9–11 . A major drawback of these methods has been the high specificity to a particular protein which requires tailoring of conditions in each case; from that point of view, a versatile chemical general tool, applicable for any target protein is yet to be developed, and is highly needed.…”