Intensified expression and purification of a recombinant biosurfactant protein

• Developed a parallel molecular and process design strategy to produce biosurfactant protein.
• No tag protein, enzymatic cleavage, mechanical cell disruption, or chromatography was required.
• Cell disruption and precipitation of contaminants achieved by thermal treatment.
• Biosurfactant expression yield of 15 mg L−1 OD600−1 in fully synthetic minimal media.
• Biosurfactant recovered by filtration and precipitation units only.

Proteins and peptides are emerging as components for novel materials that are switchable in response to their environment, and have enhanced sustainability over traditional materials. Proteins and peptides are known to be surface active and are widely used to stabilise foams and emulsions. However, designed surfactant proteins and peptides are presently produced using non-industrial processes based, for example, on costly chromatographic approaches developed for biopharmaceuticals. Here we report an intensified chromatography-free process for protein and peptide surfactant manufacture, for a recently-reported helix-bundle class of biosurfactants. The helix bundle structure is shown to remain stable and soluble under temperature and salt conditions that disrupt cells and precipitate cellular proteins. This finding opens a process route which simply involves heating cells, in fermentation media, to high temperature (e.g. 95 °C), leading to release of soluble biosurfactant with simultaneous precipitation of contaminants. This “bake-to-break and precipitate” (BPP) process allows recovery of purified biosurfactant through simple thermal treatment followed by solid–liquid separation. Experiments were conducted with the four-helix bundle protein DAMP4, which was expressed intracellularly at a level of 15 mg L−1 OD600−1 in fully synthetic minimal media. Thermal treatment of 100 mL of E. coli suspension at OD600=4 produced 4.8 mg of functional protein surfactant at a yield of 84% following simple microfiltration. Further polishing by precipitation and filtration gave 2.4 mg of highly-pure biosurfactant. This work demonstrates that co-considered molecular and intensified process design can be used to progress the development of new biological products into low-cost industrial sectors such as those based on surfactants.

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