E. coli playing the role of cobra

Moscow researchers have solved the most challenging problem: they made E. coli synthesize one of the most toxic elements of cobra’s poison. It was no simpler a task than keeping a terrarium. The scientists’ efforts were supported by the Russian Foundation for Basic Research and INTAS.

Natural poisons have always been an attraction for researchers, but it is very hard to study them as poisons are multi-componential and each of them affects cells in its own specific way. Such are alpha-neurotoxins – the most toxic component of cobra’s poison. The development of medicine to cure diseases of the nervous system requires data on the structure and properties of the alpha-neurotoxins and expertise in their behavior at the molecular level. Researchers from the Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences got interested in neurotoxin II which interacts in a specific way with a certain type of the muscular cells receptors . To investigate the effect of the toxin, it is necessary to constantly obtain the required protein – pure and sufficient in quantity. Such tasks are traditionally solved with the help of E. coli: the required gene is forced into it and made working (it is very costly, inconvenient and troublesome to get poison from a snake before each experiment). However, it is practically impossible to obtain alpha-neurotoxin II using such a method, as this protein is unable to fold into its proper shape in the bacterium cell and is very quickly destroyed. But Moscow bioorganic chemists managed to avoid these problems and to obtain a sufficient quantity of stable protein. It was actually no simpler a task than keeping a terrarium.

In order to make E. coli synthesize neurotoxin, it was necessary to obtain the gene of this toxin first. The gene was constructed out of ten DNA pieces having been synthesized in the laboratory. A gene of the E. coli natural protein – thioredoxin – was also included into the construction to stabilize neutrotoxin. After completing its task, thioredoxin should be split off from the produced protein to avoid hanging around the toxin as a redundancy. For this purpose, a special DNA part was inserted between two genes as a kind of a molecular button allowing the subsequent disposal of the redundant piece.

The scientists had to assemble the final construction in several phases and then to correct its structure. But their efforts were paid off. They received a genetically-engineered protein which in accordance with the physico-chemical analysis is identical to the natural neurotoxin. 1 liter of bacterium culture yielded about 6 mg of protein – such quantity is sufficient enough for investigating the interaction of toxin and its mutations with cell receptors. No foreign research team has ever managed to obtain such pure toxin and in such quantity though there was quite a number of attempts. The system has another advantage. If by any chance the modified E. coli ‘escapes’ the laboratory it will not pose any danger. In order to obtain a soluble toxin, bacteria should be grown at 12oC – the temperature which is uncomfortable for them – and then thioredoxin stabilizing the protein should be removed to the conditions non-existent in nature. The researchers are planning to conduct comparative biological tests for two alpha-neurotoxins II – the artificial and natural ones.