Sindarovska I. Agrobacterium-mediated transient expression of genes of recombinant proteins (GFP, beta-glucuronidase and interferon alpha 2b) in plants of Nicotiana L. genus

The thesis focuses on protocol development for recombinant protein production by Agrobacterium-mediated transient expression of foreign genes in plants. The aim of the work was to develop and optimize conditions for the production of recombinant proteins in plants by Agrobacterium-mediated transient expression. A research object was Agrobacterium-mediated transient expression in plants. Following methods were used in the work: agroinfiltration of intact plants, genetic transformation of plants, molecular biological methods, biochemical and biophysical methods of protein determination, ion-exchange chromatography and interferon titration. As a result of the work for the first time new host species for recombinant proteins production among Nicotiana genus were selected. The plant species demonstrating high content of reporter protein obtained after transient expression of transgene inserted in different types of expression cassettes were chosen (N. cavicola, N. exigua and N. excelsior). For the first time it is proved that highest content of target protein was observed in the 2nd-6th or the 2nd-4th leaves from apex (depending on expression cassettes) in adult plants before flowering. It is shown that the viral suppressor of gene silencing р19 increases content of reporter protein. For the first time it is proved that exogenous phytohormones, methyljasmonate and inhibitors of its biosynthesis did not influence on the content of target protein after transient expression. For the first time the transgenic plants of N. benthamiana with increased biomass (in vitro conditions) were obtained from "hairy roots" culture. The method of the two-steps purification of green fluorescent protein was developed. A method allows for getting 77% yield of initial amount of protein with 85% purity. For the first time transient expression of beta- glucuronidase gene in N. cavicola and N. excelsior plant was demonstrated. Also the possibility of obtaining of biologically active human protein (leukocyte interferon alpha 2b) was shown in N. excelsior plants after transient expression. The results of research could be used for production of target proteins important for medicine and pharmacology by transient expression method in plants.