Multimodal approaches for the improvement of the cellular folding of a recombinant iron regulatory protein in E. coli

Abstract

Background 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. Results The cells exposed to osmolytes plus a pre-induction heat shock showed a substantial increase in recombinant aconitase activity when combined with modulation of process conditions. 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. Conclusions 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. Graphical Abstract

Increase CO 2 recycling of Escherichia coli containing CBB genes by enhancing solubility of multiple expressed proteins from an operon through temperature reduction

“…Low culture temperature for recombinant protein expression in E. coli is a well-established strategy to suppress IB formation ( 13 ). At low temperatures, protein solubility is enhanced, and protein aggregation is reduced ( 14 ). Therefore, based on these findings, it is evident that temperature control from 37°C to 30°C effectively suppresses IB formation and enhances the endogenous CO 2 recycling capacity of the CBB strain.…”

Double promoter and tandem gene strategy for efficiently expressing recombinant FGF21

“…Images of the stained gel were captured using a Bio-Rad Molecular Imager® Gel Doc XR + Imaging Unit. The relative quantification of band intensity was estimated using Image Lab® software in the Bio-Rad Molecular Imager Gel Doc XR + unit by densitometric analysis [ 37 ].…”

The s-oph enzyme for efficient degradation of polyvinyl alcohol: soluble expression and catalytic properties

“…For example, Raziyeh Malekian et al used molecular chaperones to achieve soluble expression of GM-CSF protein in E. coli [21]; Safar Farajnia et al found that molecular chaperones have signi cant advantages in improving soluble expression of Fab antibody in E. coli [22]; Shuaiying Peng et al studied found that co-expression of PFA with molecular chaperone proteins signi cantly increased the soluble expression of PFA [23]. So we continued to combine molecular chaperones to alter the soluble expression of s-oph proteins [24].…”