| Abstract [eng] |
The principal advantages of S. cerevisiae yeast to express recombinant proteins was used to study expression peculiarities of the fused bacterial thioredoxin or glucose dehydrogenase genes with Abeta peptide sequence. In the next step of this work we investigated the growth conditions of stabily persisting yeast transformants in order to achieve a maximal production of the hybrid proteins. In the present work yeast expression vectors bearing either thioredoxin-encoding gene fused with Abeta peptide (Trx-ab) or its mutant sequence or glucose dehydrogenase gene fused with Abeta (GDH-ab) under the control of galactose-inducible GAL-CYC1 promoter were constructed. After successful transformation of yeast Saccharomyces cerevisiae strain 21PMR the eukaryotic producents of the hybrid proteins were isolated. Analysis of stability of heterologous expression plasmids indicated 90-95% maintenance of auxotrofic markers and only 50% - that of Trx-ab or GDH-ab genes. Intracellular and extracellular yeast expression system were designed to ensure synthesis of tioredoxin fused with Abeta peptide. Optimization of yeast cultivation conditions and purification procedures showed that the highest level of hybrid protein is observed in the cytoplasmic fraction of intracellular producent. Expression pecularities of the glucose dehydrogenase gene and its hybrid, bearing an additional Abeta peptide sequence, controlled by constitutive ADH1 and galactose-inducible GAL-CYC1 promoters in yeast S. cerevisiae were investigated. It was found that the optimal expression level of bacterial GDH and GDH-ab is achieved only by the using of inducible expression system. The obtained data suggest, the plasmid genetic instability of yeast transformants leads to accumulation lower quantities of the desired protein. Expression and primary purification of both Trx-ab and GDH-ab proteins is successfully performed using yeast S. cerevisiae. The yield from 1 g of yeast culture reached about 20-25 mg of the target proteins. |
