Extraction of recombinant proteins from Escherichia coli by cell disruption with aqueous solutions of surface‐active compounds

Abstract

BACKGROUND: Green fluorescent protein (GFP) is used extensively as a biomarker due to its unique spectral and fluorescence characteristics. GFP is usually obtained from recombinant strains of Escherichia coli (E. coli) producing the protein intracellularly. However, the common methods of extraction are cumbersome leading to an increase in the downstream process costs and complexity, sometimes leading to a higher risk of biomolecule degradation. RESULTS: This work proposes a new method to extract recombinant intracellular GFP from E. coli BL21 by using aqueous solutions of surface-active compounds.CONCLUSION: By comparing the fluorescence intensity of the extracted GFP, it was concluded that some of these compounds, namely ionic liquids with an alkyl chain of 10 or more carbons (best solvent being tributyl-1-tetradecylphosphonium, [P 4,4,4,14 ]Cl) are more effective than an ultrasonic-assisted extraction, even at low concentrations, being able to extract the whole GFP content from the cells. Associated Content Supporting information. General information of the surface-active compounds structures used, as well as their CMCs, and experimental results on the cell viability after GFP release using the best surface-active compounds.

Extraction and purification of violacein from Yarrowia lipolytica cells using aqueous solutions of surfactants

“…show that the non‐tensioactive ILs were unable to permeate the cell membrane, thus being ineffective in violacein release. This is probably due to the low ability of hydrophilic ILs to disrupt cell membranes, considering their low affinity to interact with the phospholipids composing the membranes, due to their hydrophobic nature . On the other hand, and as expected, most of the surfactant compounds, particularly the non‐ionic surfactants, allowed the disruption of cell membranes, leading to violacein release to a larger extent than the control solvents.…”

“…The common surfactants tested in this work were pre‐selected considering some works previously reported by us, where, from a large list, these were considered as the most efficient on cell disruption . The results presented in Fig.…”

“…The chemical method for cell disruption was adapted from the literature . The cell suspension was homogenized with aqueous solutions of surfactants to test their ability to release violacein.…”

Synthesis and Characterization of Surface‐Active Ionic Liquids Used in the Disruption of Escherichia Coli Cells

“…Moreover, the experimental data obtained also suggest that similar results (for some SAILS differing only in 5 %) can be obtained by the application of aqueous solutions of tensioactive compounds when compared with the ultrasonication disruptive performance. In addition to the similar disruptive performance, the use of aqueous solutions of SAILs instead of mechanical treatments is normally more sustainable, since the energy costs are decreased …”

“…The GFP extraction from E. coli BL21 cells by using tensioactive compounds was recently proposed by our group as an alternative method to conventional ultrasonic‐assisted extraction. The preparation of the cell culture and the GFP release (cell disruption) was performed according to previous work . Briefly, the E. coli strain was grown at 310.2 K in LB medium with kanamycin and chloramphenicol (50 μg mL −1 ) with a constant stirring (150 rpm).…”