Ations. Living cells can support the stability of proteins by a

Ations. Living cells can support the stability of proteins by a number of organic substances known also as chemical chaperones [2]. Upon recombinant protein production, such chemicals are unfortunately only of limited value as access to the inner cell compartment in conventional cell-based expression systems is restricted. Increasing intracellular concentrations of stabilizers by e.g. inducing specific solute transporters requires strong impacts such as osmotic shocks which could cause dramatic changes in cell physiology and expression patterns [3,4]. Stabilization strategies are therefore usually confined to manipulations of growth conditions or to attempts of post-translational stabilization during protein extraction, when significant protein precipitation might already have occurred. Cell-free (CF) expression systems offer the new option to support the stability of expressed proteins already co-translation-ally with 1326631 a wide and diverse range of additives, while on the other hand being Dimethylenastron biological activity relatively sensitive to manipulations of reaction conditions such as incubation temperature. The open nature of CF reactions allows to supply any tolerated chemical directly into the protein expression environment [5]. Production protocols for unstable and difficult proteins can therefore be individually designed and stabilizers or mixtures thereof can be adjusted according to specific requirements. Protein stabilizing agents comprise a wide range of chemicals including alcohols and molecular crowding agents such as polyethylenglycols (PEG). Many organisms accumulate small organic molecules in stress situations, which are generally called osmolytes [6,7]. Those solutes act as chemical chaperones in the cell by preventing protein unfolding and improving protein thermostability. Major groups of osmolytes are polyols, amino acids, polyions or urea [2]. Prominent examples are the synthesis of betaine or trehalose in E. coli, glycerol in Saccharomyces cerevisiae and generally a number of different polyols and amino acid derivatives in yeasts and plants [7]. Hyperthermophilic microorganisms accumulate organic solutes such as betaine, ectoine or trehalose in high concentrations while responding to heat stress [8,9]. The intracellular concentration of some of these compounds can even reach molar levels dependent on medium osmolality and growth conditions [10].Chemical Chaperones for Improving Protein QualityCF reactions are ideal for screening experiments and have been applied for the expression of target libraries [11?3], protein evolution [14] or drug screening [15]. We have established a process based on extracts of E. coli cells and on the batch configuration that allows the screening of chemical chaperones. The tolerated concentration ranges of all additives 1326631 were determined in linear screening schemes and by using shifted green fluorescent protein (sGFP) as expression monitor. Additives showing positive effects on sGFP fluorescence were then further analyzed in linear or in correlated screening schemes for their effects on two unstable proteins. The screening process for cotranslational protein stabilization was exemplified with the human glucosamine 6-phosphate N-acetyltransferase (GNA1) and with the 4EGI-1 web halogenase domain of the fungal CurA polyketide synthetase [16]. Improved solubility of the two proteins was in particular monitored with choline and L-arginine and cumulative effects of selected compounds were analyzed in correlated screens. The established p.Ations. Living cells can support the stability of proteins by a number of organic substances known also as chemical chaperones [2]. Upon recombinant protein production, such chemicals are unfortunately only of limited value as access to the inner cell compartment in conventional cell-based expression systems is restricted. Increasing intracellular concentrations of stabilizers by e.g. inducing specific solute transporters requires strong impacts such as osmotic shocks which could cause dramatic changes in cell physiology and expression patterns [3,4]. Stabilization strategies are therefore usually confined to manipulations of growth conditions or to attempts of post-translational stabilization during protein extraction, when significant protein precipitation might already have occurred. Cell-free (CF) expression systems offer the new option to support the stability of expressed proteins already co-translation-ally with 1326631 a wide and diverse range of additives, while on the other hand being relatively sensitive to manipulations of reaction conditions such as incubation temperature. The open nature of CF reactions allows to supply any tolerated chemical directly into the protein expression environment [5]. Production protocols for unstable and difficult proteins can therefore be individually designed and stabilizers or mixtures thereof can be adjusted according to specific requirements. Protein stabilizing agents comprise a wide range of chemicals including alcohols and molecular crowding agents such as polyethylenglycols (PEG). Many organisms accumulate small organic molecules in stress situations, which are generally called osmolytes [6,7]. Those solutes act as chemical chaperones in the cell by preventing protein unfolding and improving protein thermostability. Major groups of osmolytes are polyols, amino acids, polyions or urea [2]. Prominent examples are the synthesis of betaine or trehalose in E. coli, glycerol in Saccharomyces cerevisiae and generally a number of different polyols and amino acid derivatives in yeasts and plants [7]. Hyperthermophilic microorganisms accumulate organic solutes such as betaine, ectoine or trehalose in high concentrations while responding to heat stress [8,9]. The intracellular concentration of some of these compounds can even reach molar levels dependent on medium osmolality and growth conditions [10].Chemical Chaperones for Improving Protein QualityCF reactions are ideal for screening experiments and have been applied for the expression of target libraries [11?3], protein evolution [14] or drug screening [15]. We have established a process based on extracts of E. coli cells and on the batch configuration that allows the screening of chemical chaperones. The tolerated concentration ranges of all additives 1326631 were determined in linear screening schemes and by using shifted green fluorescent protein (sGFP) as expression monitor. Additives showing positive effects on sGFP fluorescence were then further analyzed in linear or in correlated screening schemes for their effects on two unstable proteins. The screening process for cotranslational protein stabilization was exemplified with the human glucosamine 6-phosphate N-acetyltransferase (GNA1) and with the halogenase domain of the fungal CurA polyketide synthetase [16]. Improved solubility of the two proteins was in particular monitored with choline and L-arginine and cumulative effects of selected compounds were analyzed in correlated screens. The established p.