Small potent inhibitors of aggregation are eagerly demanded for preventing the inactivation of proteins. This paper shows that amino acid esters (AAEs) prevent heat-induced aggregation and inactivation of hen egg lysozyme. Lysozyme was completely inactivated (<1% original activity) during heat treatment at 98 degrees C for 30 min in a solution containing 0.2 mg/mL lysozyme in 50 mM Na-phosphate buffer (pH 6.5). The residual activities only slightly increased (<5%) in the presence of 100 mM commonly used additives such as arginine, guanidine, urea, and sugars. However, in the presence of 100 mM AAEs, the residual activities were >60% and no aggregates were observed during the heat treatment at 98 degrees C for 30 min. This fact provides new information on the scaffold for designing additives to prevent heat-induced aggregation.
Misfolding poses a serious problem in the biotechnological field in obtaining the active protein from inclusion bodies. Here we show that high temperature increases the refolding yield of reduced lyosyzme by a simple dilution method. The refolding yields at 98 degrees C were three times higher than those at 20 degrees C in the solutions tested, which is related to the fact that the thermally unfolded state of lysozyme is a more productive form for folding than the denaturant-induced fully unfolded state. The thermal-assisted refolding could be used for various reduced and denatured proteins as a result of its simplicity and low cost.
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