Optimized release of recombinant proteins by ultrasonication ofE. coli cells

Abstract

The release kinetics of ␤-galactosidase protein have been determined during small-scale ultrasonication of E. coli cells. Among several studied parameters, ionic strength and cell concentration have the least influence on the rate of protein recovery, whereas sample volume and acoustic power dramatically affect the final yield of soluble protein in the cell-free fraction. The analysis of these critical parameters has prompted us to propose a simple model for E. coli disintegration that only involves acoustic power and sample volume, and which allows prediction of optimal sonication times to recover significant amounts of both natural and recombinant proteins in a given set of relevant conditions.

Divergent Genetic Control of Protein Solubility and Conformational Quality in Escherichia coli

“…1 836 170) per 10 ml of buffer. These samples, once jacketed in ice, were sonicated for 5 min (or longer when required to achieve a complete disruption) at 50 W under 0.5 s cycles as described, 69 and centrifuged for 15 min at 15,000g. The supernatant was mixed with denaturing buffer at the appropriate ratios.…”

“…Culture samples of 1 ml were jacketed in ice, disrupted by sonication for 4 min at 50 W under 0.5 s cycles as described, 69 and centrifuged at 4°C for 15 min at 15,000g. The supernatant was used directly for the analysis as the soluble cell fraction.…”

Enzymatic production of β‐D‐glucose‐1‐phosphate from trehalose

“…Expression host cells can be disrupted completely to release recombinant enzymes and perform reactions of interest with cell-free extracts. These methods range from mechanical such as ultrasonication [24] to enzymatic through the use of lysozyme [25] to chemical methods by using detergents or EDTA and urea [26]. A significant advantage of the use of thermostable enzymes is the possibility to use the exposure to higher temperatures to achieve a similar effect.…”

Microalgal cell disruption by hydrodynamic cavitation for the production of biofuels

“…The generation of cavitation can be ultrasonic (Feliu et al 1998), optic (Vogel and Lauterborn 1988) or hydrodynamic (Yu et al 1995). For liquids with high boiling point and low viscosity, the collapse of these cavities can be very violent with extremely localised zones of pressure as high as 100 MPa and temperature up to 5000°C (Gogate and Pandit 2005).…”