Efficient Refolding of Aggregation-prone Citrate Synthase by Polyol Osmolytes

Abstract

Efficient refolding of proteins and prevention of their aggregation during folding are of vital importance in recombinant protein production and in finding cures for several diseases. We have used citrate synthase (CS) as a model to understand the mechanism of aggregation during refolding and its prevention using several known structure-stabilizing cosolvent additives of the polyol series. Interestingly, no parallel correlation between the folding effect and the general stabilizing effect exerted by polyols was observed. Although increasing concentrations of polyols increased protein stability in general, the refolding yields for CS decreased at higher polyol concentrations, with erythritol reducing the folding yields at all concentrations tested. Among the various polyols used, glycerol was the most effective in enhancing the CS refolding yield, and a complete recovery of enzymatic activity was obtained at 7 M glycerol and 10 g/ml protein, a result superior to the action of the molecular chaperones GroEL and GroES in vitro. A good correlation between the refolding yields and the suppression of protein aggregation by glycerol was observed, with no aggregation detected at 7 M. The polyols prevented the aggregation of CS depending on the number of hydroxyl groups in them. Stopped-flow fluorescence kinetics experiments suggested that polyols, including glycerol, act very early in the refolding process, as no fast and slow phases were detectable. The results conclusively demonstrate that both the thermodynamic and kinetic aspects are critical in the folding process and that all structure-stabilizing molecules need not always help in productive folding to the native state. These findings are important for the rational design of small molecules for efficient refolding of various aggregation-prone proteins of commercial and medical relevance.Aggregation of proteins during folding, both in vitro and in vivo, is known to lead to low native protein yields as well as the onset of several age-related diseases (1). Hence, there is a growing interest in developing strategies to prevent protein aggregation to enhance protein refolding yields and to design drugs for diseases involving protein aggregation. Several attempts have been made in this direction with successes as well as failures (2-4). In this study, we used citrate synthase (CS), 1 a non-disulfide-bonded dimeric protein (ϳ100 kDa) highly prone to aggregation during in vitro refolding (5-12), as a model protein. The aggregation-prone nature of CS during its refolding has made it an attractive model system to study the effect of molecular chaperones on the prevention of its aggregation and to develop strategies for enhancing protein refolding yields (8,(13)(14)(15)(16)(17)(18)(19)(20). Using the molecular chaperones GroEL and GroES, up to 80% refolding of CS could be obtained at 10 g/ml protein, whereas unassisted refolding was only 5% (7). Inspired by the GroEL/ES-assisted two-step folding mechanism, Rozema and Gellman (9) proposed an artificial chaperone-assisted ref...

In vivo detection and quantification of chemicals that enhance protein stability

“…Consistent with our in vivo results, they found that glycerol and sorbitol were the most effective as osmolytes, with 2 M and 7 M glycerol enhancing CS refolding yield 1.5-and 2.25-fold, respectively. Sorbitol at 1 M was also effective, enhancing CS refolding yield 1.5-fold [10]. Like us, they also observed that ethylene glycol and erythritol were less effective as osmolytes than glycerol or sorbitol.…”

“…Further, Mishra et al observed a very poor correlation between osmolyte concentration and refolding yield [10]. Higher concentrations of osmolytes in general were found to actually decrease citrate synthase refolding yields and, as noted, some osmolytes like erythritol even decreased folding yields at all concentrations tested.…”

“…Mishra et al 's., in vitro study [10], however, used the same conditions and compared the ability of 5 different chemical additives (glycerol, sorbitol, xylitol, ethylene glycol, and erythritol) to enhance citrate synthase (CS) refolding yield at 25 °C. Consistent with our in vivo results, they found that glycerol and sorbitol were the most effective as osmolytes, with 2 M and 7 M glycerol enhancing CS refolding yield 1.5-and 2.25-fold, respectively.…”

“…They act to maintain cell volume and stabilize macromolecules under conditions of environmental stress like high osmotic and salt pressure [7; 8; 9]. Subsequent studies showed that these compounds stabilize proteins in vitro and assist in their refolding [10]. Intense efforts have also resulted in the discovery of compounds termed pharmacological chaperones that specifically stabilize disease related mutant proteins [11; 12].…”

Structural basis of jasmonate-amido synthetase FIN219 in complex with glutathione S-transferase FIP1 during the JA signal regulation

“…The expression vector was transformed into E. coli BL21(DE3) cells, grown at 37°C in LB medium until the optical density achieved 12 at 600 nm. The 3% (vol/vol) ethanol was added during expression for inducing the endogenous chaperones of E. coli to assist the recombinant protein folding (48). The expression of GST-FIN219 proteins was induced by 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 18 h at 16°C.…”