Identification, quantitation and purification of insecticidal pr

Plastic article or earthenware shaping or treating: apparatus – Product or preform repair or restoring means – Tire recapping – rebeading or sidewall replacing means

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435 30, 435 18, 435 4, 435 681, 43525231, 4352525, 4353171, C12Q 137, C12Q 124, C12Q 100, C12N 120

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055232116

ABSTRACT:
A process for identifying proteinaceous protoxins expressed by Bacillus thuringiensis genes is disclosed. According to the process, daughter toxins are first generated by subjecting a protoxin-containing material, such as parasporal crystals of Bacillus thuringiensis, to limited proteolysis with a proteolytic enzyme in an aqueous suspension having a pH above 9.5. The daughter toxins are then separated by high performance anion-exchange liquid chromatography at a constant pH in excess of 10 in an increasing gradient of a salt, preferably sodium chloride. The gradient conditions, which are specific for the column used, are achieved by employing a series of buffers having increasing concentration of the salt and introduced at a predetermined time and rate. The procedure provides a chromatogram showing clearly identifiable peaks of toxins and permits therefore the qualitative and quantitative characterization of the original mixture and isolation of the individual toxins. By this it provides a way of screening and testing new Bacillus thuringiensis isolates, both single--and multigene, and monitoring the level of expression of known genes from a known strain. The digestion and isolation conditions permit the production of the toxins in a biologically fully active state.

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