Application Note: High Pressure Homogenisation and Protein Expression

by in Application Notes

Inspiralis is a company based in Norwich which specialises in providing topoisomerases, ligases and other DNA-modifying proteins which are used in the drug-discovery process by the pharmaceutical industry and academia. Topoisomerases are proteins which alter the topology of DNA, preventing it becoming tangled, an inherent risk when separating the two strands of the double-helix during transcription, replication and other cellular processes. These amazing molecular machines are able to do this by performing the apparent ‘trick’ of passing one piece of DNA through another and resealing it. They can be found in all organisms including eukaryotes, bacteria and viruses. Since their mechanism of action involves the transient cleavage of one or both strands of the DNA, stalling the enzymes at this point leads to potentially cytotoxic events. As a result, they are excellent targets for both antibiotics and anti-cancer compounds. Drugs such as ciprofloxacin and camptothecin have been successfully used in anti-bacterial and anti-cancer therapy respectively and they remain of great interest for researchers looking for novel drugs in these areas.

Inspiralis produces many of these proteins by over-expression in Escherichia coli (e-coli) cells and then purifying them from the extracts prepared from these bacteria. The preparation of these extracts is a key part of the purification process. This has typically been performed within Inspiralis using a French Press of some vintage. However, we have recently switched to using an Avestin Emulsiflex [high pressure homogeniser]. We needed a machine which would efficiently break the cells open whether we are using small trial samples or starting from litres of overexpressing cultures but which wouldn’t occupy a large amount of space. The Emulsiflex performs these functions perfectly.

Dr Nicolas Burton

Director & Co-Founder of Inspiralis Ltd.

Norwich BioIncubator, Norwich Research Park

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