Shunsuke Tomita

Arginine controls heat-induced cluster-cluster aggregation of lysozyme at around the isoelectric point.

The process of protein aggregation has attracted a great deal of research attention, as aggregates are first of all a nuisance to preparation of high quality protein and secondly used as novel materials. In the latter case, the process of protein aggregation needs to be controlled. Here, we show how arginine (Arg) regulates the process of heat-induced protein aggregation. Dynamic light scattering and transmission electron microscopy revealed that heat-induced aggregation of lysozyme at around the isoelectric point occurred in a two-step process: formation of start aggregates, followed by further growth mediated by their sticking with diffusion-limited cluster-cluster aggregation. In the presence of Arg, the diffusion-limited regime changed to reaction-limited cluster-cluster aggregation. The data indicated that the solution additives that coexisted with proteins would affect the property of the formed product, such as morphology and mechanic strength.

Enzyme switch by complementary polymer pair system (CPPS)

Manipulation of enzyme activity at will presents various research and industrial applications. Herein we describe development of a technology for inactivation and reactivation of enzyme activities using a polyanionic poly(acrylic acid) (PAAc) and a polycationic poly(allylamine) (PAA). Enzyme activities of ribonuclease A (RNase A), lysozyme, cellulase, and α-amylase were lost through addition of PAAc or PAA because of their binding to the enzymes. The activity of these enzymes except for α-amylase was then fully restored from the complex by the addition of oppositely charged polymers. Such manipulation of enzyme activity using a complementary polymer pair system (CPPS) enables the expansion of biomedical and biotechnological applications of the enzymes, including realization of protein delivery and intelligent bioreactors.

Why do solution additives suppress the heat-induced inactivation of proteins? Inhibition of chemical modifications

Thermoinactivation of proteins is prevented by several kinds of solution additives such as chaotropes, amino acids, amino acid derivatives, and polyamines. Here, we investigated the molecular mechanisms of action of the various additives that prevent thermoinactivation of bovine pancreatic ribonuclease A and hen egg white lysozyme. The thermoinactivation of both proteins in the presence of additives showed clear correlations with deamidation and β‐elimination of the proteins. Thus, experimental evidences indicated that the effects of additives on thermoinactivation of proteins are highly due to the suppression of chemical modifications. To our surprise, not only the suppressive effect of the additives on heat‐induced inactivation but also that on the chemical modification of proteins is remarkably similar by comparison of two unrelated proteins. This finding indicates the generality of the effects of additives on heat‐induced chemical modification of proteins.

Different mechanisms of action of poly(ethylene glycol) and arginine on thermal inactivation of lysozyme and ribonuclease A

Proteins tend to undergo irreversible inactivation through several chemical modifications, which is a serious problem in various fields. We have recently found that arginine (Arg) suppresses heat‐induced deamidation and β‐elimination, resulting in the suppression of thermal inactivation of hen egg white lysozyme and bovine pancreas ribonuclease A. Here, we report that poly(ethylene glycol) (PEG) with molecular weight 1,000 acts as a thermoinactivation suppressor for both proteins, especially at higher protein concentrations, while Arg was not effective at higher protein concentrations. This difference suggests that PEG, but not Arg, effectively inhibited intermolecular disulfide exchange among thermally denatured proteins. Investigation of the effects of various polymers including PEG with different molecular weight, poly(vinylpyrolidone) (PVP), and poly(vinyl alchol) on thermoinactivation of proteins, circular dichroism, solution viscosity, and the solubility of reduced and S‐carboxy‐methylated lysozyme indicated that amphiphilic PEG and PVP inhibit intermolecular collision of thermally denatured proteins by preferential interaction with thermally denatured proteins, resulting in the inhibition of intermolecular disulfide exchange. These findings regarding the different mechanisms of the effects of amphiphilic polymers––PEG and PVP––and Arg would expand the capabilities of methods to improve the chemical stability of proteins in solution. Biotechnol. Bioeng. 2012; 109: 2543–2552.

Improving the Heat Resistance of Ribonuclease A by the Addition of Poly(N,N-diethylaminoethyl methacrylate)-graft-poly(ethylene glycol) (PEAMA-g-PEG)

Poly(N,N-diethylaminoethyl methacrylate)-graft-poly(ethylene glycol) (PEAMA-g-PEG) has previously been used as a novel additive to improve the heat resistance of lysozyme, which has a positive net charge and a negatively charged active site. In the present study, we show that PEAMA-g-PEG prevents heat inactivation of ribonuclease A (RNase A), which has a positive net charge and a positively charged active site. After treatment at 98u2009°C for 10u2009min, the enzymatic activity of RNase A complexed with PEAMA-g-PEG was maintained at up to 75% of the level of the native RNase A. The extents of inactivation of RNase A and the complex of RNase A with PEAMA-g-PEG were strongly dependent upon the heating temperature and incubation time. Circular dichroism (CD) spectral analysis revealed that heat-induced irreversible inactivation was largely suppressed when RNase A was complexed with PEAMA-g-PEG. These findings suggest that the heat resistance of RNase A is improved by the external addition of PEAMA-g-PEG.

Glutathione Ethylester, a Novel Protein Refolding Reagent, Enhances both the Efficiency of Refolding and Correct Disulfide Formation

Protein refolding constitutes a crucial process for recombinant proteins. We report here on the development of a multifunctional refolding additive, glutathione ethyl ester (GSHEE), prepared from a redox reagent glutathione and an amino acid ethyl ester, an aggregation suppressor. Compared to glutathione, GSHEE showed 3.2-fold higher efficiency for the refolding yield of hen egg lysozyme. More importantly, a low concentration of GSHEE is more effective for refolding than conventional additives, such as amino acid ethyl esters by two orders of magnitude. The high potency of GSHEE makes it a candidate for use as a refolding additive for use in conjunction with reduced and denatured proteins.

Synergistic effect of polyethylene glycol with arginine on the prevention of heat-induced aggregation of lysozyme

Abstract. Arginine (Arg) is a commonly used additive to prevent protein aggregation and inactivation in denaturing solutions. This paper presents new findings on the synergistic effect on the prevention of heat-induced aggregation of lysozyme using Arg in combination with polyethylene glycol (PEG). The synergistic enhancement was observed in the presence of Arg with amphiphilic polymers, such as PEG6000, PEG20000, and poly(vinyl pyrrolidone), while it was not observed with hydrophilic polymers, such as PEG200, Poly(acrylic acid), poly(vinyl alcohol), dextran, and Ficoll 70.