Multimodal Approaches for the Improvement of the Cellular Folding of a Recombinant Iron Regulatory Protein in E. Coli

Abstract

BackgroundDuring the recombinant protein expression, foreign proteins are generated in insoluble and inactive aggregates in E. coli cell factories, which inhibits 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 process variables such as incubation temperature, inducer concentrations, growth media, and the presence of folding modulators such as exogenous molecular chaperones or osmolytes are crucial for the cellular folding and are investigated in the study. The processes the physiological stress such as osmotic and heat shock stimulation in the host cells and thereby their effect on the solubility and activity of recombinant proteins was also analysed.ResultsOf the various methods discussed, the cells subjected to the addition of osmolytes and pre-induction heat shock exhibited significant enhancement in the recombinant aconitase activity. The concomitant GroEL/ES expression further assists the folding of these soluble aggregates and increases the functional protein molecules in the cytoplasm of the recombinant E. coli cells.ConclusionsThe 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 productivity 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.