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Cellular Osmolytes: From Chaperoning Protein Fo... ((HOT))


Mutations in the cystic fibrosis transmembrane conductance regulator protein (CFTR) often result in a failure of the protein to be properly processed at the level of the endoplasmic reticulum (ER) and subsequently transported to the plasma membrane. The folding defect associated with the most common CFTR mutation (delta F508) has been shown to be temperature sensitive. Incubation of cells expressing delta F508 CFTR at lower growth temperatures results in the proper processing of a portion of the mutant CFTR protein. Under these conditions, the mutant protein can move to the plasma membrane where it functions, similar to the wild-type protein, in mediating chloride transport. We set out to identify other methods, which like temperature treatment, would rescue the folding defect associated with the delta F508 CFTR mutation. Here we show that treatment of cells expressing the delta F508 mutant with a number of low molecular weight compounds, all known to stabilize proteins in their native conformation, results in the correct processing of the mutant CFTR protein and its deposition at the plasma membrane. Such compounds included the cellular osmolytes glycerol and trimethylamine N-oxide, as well as deuterated water. Treatment of the delta F508 CFTR-expressing cells with any one of these compounds, which we now refer to as 'chemical chaperones', restored the ability of the mutant cells to exhibit forskolin-dependent chloride transport, similar to that observed for the cells expressing the wild-type CFTR protein. We suggest that the use of 'chemical chaperones' may prove to be effective for the treatment of cystic fibrosis, as well as other genetic diseases whose underlying basis involves defective protein folding and/or a failure in normal protein trafficking events.




Cellular Osmolytes: From Chaperoning Protein Fo...


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During the recombinant protein expression, most heterologous proteins expressed in E. coli cell factories are generated as insoluble and inactive aggregates, which prohibit E. coli from being employed as an expression host despite its numerous advantages and ease of use. The yeast mitochondrial aconitase protein, which has a tendency to aggregate when expressed in E. coli cells in the absence of heterologous chaperones GroEL/ES was utilised as a model to investigate how the modulation of physiological stimuli in the host cell can increase protein solubility. The presence of folding modulators such as exogenous molecular chaperones or osmolytes, as well as process variables such as incubation temperature, inducer concentrations, growth media are all important for cellular folding and are investigated in this study. This study also investigated how the cell's stress response system activates and protects the proteins from aggregation.


The recombinant E. coli cells enduring physiological stress provide a cytosolic environment for the enhancement in the solubility and activity of the recombinant proteins. GroEL/ES-expressing cells not only aided in the folding of recombinant proteins, but also had an effect on the physiology of the expression host. The improvement in the specific growth rate and aconitase production during chaperone GroEL/ES co-expression is attributed to the reduction in overall cellular stress caused by the expression host's aggregation-prone recombinant protein expression.


In recombinant cells, the high translational rates and cellular crowding due to protein over-expression together with the reducing cellular environment leads to protein misfolding. The genetic manipulations also influence the physiological characteristics of the cellular functions challenging the recombinant cells with increased metabolic burden and cellular stress that also promotes protein aggregation and significantly impedes the growth [5]. E. coli overcomes the aforesaid hurdles by activating its innate defensive systems in response to diverse physiological stresses. The common intrinsic recovery mechanism involves expression of specific sigma factors. These are tiny proteins that bind to RNA polymerase and direct them to bind specific promoters and induce expression of heat shock proteins like DnaK-DnaJ-GrpE and GroEL/ES [6, 7]. These proteins function by facilitating the refolding of aggregated proteins. The heat shock proteins, which are also called as the molecular chaperones, are the giant molecular assemblies which interact with individual misfolded proteins to assist in its folding for the attainment of the native structure [8]. GroEL/ES is one of the prokaryotic chaperone machinery comprising of the chaperonins GroEL and its co-chaperone GroES, which assists the folding of the misfolded proteins by providing a cage-like enclosure away from the crowded cellular environment [9]. However, the physiological concentrations of molecular chaperones in host organisms are insufficient to accommodate the enormous amount of misfolded recombinant proteins produced. Hence, the co-expression of higher amounts of molecular chaperones from exogenous plasmids regulated by a strong promoter must help in folding the misfolded polypeptides and improve the solubility of the proteins [10].


Modulation of environmental conditions such as temperature, inducer concentration and the medium are known to affect the protein solubility [11]. The cells grown under physiological stress conditions including osmotic and heat-shock stresses adapt to the environment to maintain normal cellular function. In order to overcome physiological stresses, the bacterial system adapts itself to synthesize bio-molecules and endogenous heat-shock proteins that can evade the harsh effects and help in normal functioning of the cells. It is now known that certain stresses elicit similar global stress responses, and that adaptation to one form of stress can lead to resistance to another [12]. In this work, we have exploited these features of the bacterial cells grown under stress conditions to improve the solubility and activity of the aggregation-prone recombinant protein. It has also been a focus of research to investigate how cells endure osmotic stress and preserve cell homeostasis by creating osmolytes, which help proteins fold properly by stabilising their structure. We used a bench-scale screening platform to optimise the productivity, solubility, and activity of recombinant yeast mitochondrial aconitase, through application of GroEL/ES assisted folding process in E. coli. The mature yeast mitochondrial aconitase is an 82 kDa monomeric metalloenzyme with Fe4S4 cluster as the prosthetic group. It catalyzes one of the tricarboxylic acid cycle reactions, the isomerization of citrate to iso-citrate in the presence of Fe4S4 acting as the prosthetic group. In this study, aconitase was taken as the model protein as it is an obligate substrate for GroEL/ES chaperone and tends to aggregate when expressed in E. coli in absence of GroEL/ES [13]. Although, the role of GroEL/ES in improving the solubility of recombinant protein has been reported elsewhere, in the present work we have not only shown the co-expressed chaperone assisted improvement of recombinant protein folding, but also demonstrated how the cellular folding of recombinant aconitase has been improved through combinatorial effect of chaperone co-expression, use of osmolytes, and induced cellular stress response.


A 2.4 kb mature yeast mitochondrial aconitase (aco1) gene was amplified from the template plasmid pQE60Aco by polymerase chain reaction and cloned downstream of the T7lac promoter in pET29 vector and the resulting plasmid was designated as pETAco. The insertion of the mature aconitase gene was confirmed by restriction digestion and gene sequencing (Additional file 1: Fig S1). Both results confirmed the cloning of the mature aconitase gene sequence in the pET29 vector. The total aconitase protein expression was checked on SDS-PAGE gel of the BL21(DE3) cells transformed with the plasmid pETAco, grown in LB media until mid-log phase and induced with 0.5 mM IPTG (Fig. 1A). The cells expressing aconitase were re-transformed with pGro7 for expression of GroEL/ES. The co-expression of both the yeast mitochondrial aconitase and GroEL/ES was verified on a 12% SDS-PAGE gel of total cell lysate from transformed BL21(DE3) cells induced with 0.5 mM of sterile IPTG (for expression of aconitase) and 0.5 g/L of l-arabinose (for expression of chaperones GroEL/ES) (Fig. 1B).


Figure 6A and B depicts the aconitase yield obtained under different temperatures and varying inducer concentrations. A rapid increase in expression levels is observed in cells induced at 37 C leading to large protein accumulation within 2 h of induction. Cells grown at 25 C on the other hand exhibit a slower rate of production but the accumulation extended over a period of 10 h in the induction regime providing the nascent polypeptides enough time to fold in the complex cellular environment. It was seen that cells induced with high IPTG concentration (2 mM), showed a very high rate of synthesis during initial phase of induction at both the temperatures which retarded after 4 h.


The co-expression of chaperones enables folding of aconitase but the expression of additional gene from an additional plasmid exerts further imbalance in the cellular metabolism and the proteins expressed compete for the tRNA pool of the cellular systems. This part of the experiments was carried out to demonstrate the effect of chaperone co-expression on the expression of the recombinant protein. The co-transformed E. coli cells were grown at 25 C till OD600 of 0.6 to 0.8 and simultaneously induced with IPTG (0.5 mM) and varying arabinose concentrations ranging from 0.01 to 0.75 mg/mL for GroEL/ES expression to study its effect on aconitase expression, solubility and activity.


The effect of media components on the biomass profiles of the recombinant BL21(DE3) cells expressing recombinant proteins. A Denotes the cellular density profiles of the cells expressing only aconitase. B Denotes the cellular density profiles of the cells expressing aconitase and GroEL/ES simultaneously. The open squares, circles, triangles and inverted triangles indicate the recombinant cells grown at 25 C in defined media, Luria broth, Yeast-Tryptone media and Terrific Broth respectively. C Depicts aconitase expression in mg/g DCW when grown in different types of media. D, E Depict the effect of media components on aconitase activity and aconitase solubility during the absence and presence of exogenous GroEL/ES expression 041b061a72


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