METHOD OF DETERMINATION OF CONDITIONS OF CRYSTALISATION OF PROTEINS Russian patent published in 2017 - IPC A23J3/00 C30B7/00 

FIELD: chemistry.

SUBSTANCE: method of crystallisation of proteins involves preparation of the initial solutions of the protein in the buffer, filtration of the resulting solution, centrifugation and filling with a solution of capillaries. The first part of the protein solutions obtained after centrifugation is mixed with the buffer solution in equal volumes, and the remaining second part is subsequently used for mixing with the precipitant. The first part of the protein solutions is analysed by small-angle of X-ray scattering to obtain a series of scattering curves, the curves are analysed and the size of a single protein subunit in each of the solutions of different concentrations is calculated. The mother liquors of the precipitant are prepared, for which substances that can influence the conformation of the molecules can be used; they filter the mother liquor of the precipitator, mix the second part of the protein solutions in equal volumes with the mother liquors of the precipitant, obtaining a collection of crystallisation solutions. Each of the solutions of the named collection is studied by small-angle of X-ray scattering in the temperature range from the temperature at which the molecule of this protein does not become denatured to 0°C with the chosen temperature change step, resulting in the graphs of the small-angle scattering curves, mathematically process these scattering curves, obtaining the distribution of the number of protein subunits and their oligomers in solution, depending on their radius, those crystallization solutions are selected, when processing the small-angle scattering curves from which the formation of the largest number of oligomers in solution was detected, which means finding conditions for crystallisation. If particles that are larger in size than the monomers are not detected in the crystallisation solutions, then step-by-step lower the temperature of the solutions under study to 0°C, at each of the stages of decreasing the temperature of the solution, each of the solutions is studied by small-angle scattering, mathematically processed by these scattering curves, obtaining the distribution of the number of particles in the solution as a function of their radius, those crystallisation solutions are selected, when processing small-angle scattering curves from which the formation of the largest number of oligomers was detected in the solution, the stepwise temperature reduction operation is completed by detecting the greatest number of oligomers, which means finding crystallisation conditions.

EFFECT: invention allows to shorten the time for the formation of crystals, as well as to create a direct method for determining the formation of growth units, providing reliable and timely information about the initial stage of crystallisation of the protein.

9 cl, 6 dwg