Abstract [eng] |
The recombinant expression of proteins in E.coli cytoplasm at high concentrations often results in the formation of insoluble aggregates known as inclusion bodies. To obtain a soluble and biologically active protein, IBs must be solubilized and refolded. The proper folding of target protein in vitro competes with unproductive side reaction such as misfolding and aggregation. To prevent aggregation and increase refolding yield of correctly folded protein, low molecular weight additives are used. A great number of articles on the refolding strategies of various proteins in the presence of different solution additives have encouraged us to test these additives on two model proteins: recombinant mink and porcine growth hormones, and provide some evidence for this refolding strategy. The refolding of recombinant growth hormones from E. coli inclusion bodies at high protein concentration and pH 8.0 was impossible in the absence of the solution additives, due to a high level of protein aggregation and precipitation. Low molecular weight additives are mainly grouped into two groups: protein stabilizers and denaturants. However, there are solution additives, which are not placed in these two groups. They have only marginal effect on protein structure and stability, but appear capable to suppress the aggregation of protein. The group of aggregation suppressors includes such solution additives as L-arginine, L-arginine ethyl ester and cyclodextrins. L-arginine and L-arginine ethyl ester effectively suppressed the precipitation of growth hormones during refolding, but they did not inhibit soluble oligomers formation. Methyl-beta-cyclodextrin and 2-hydroxypropyl-beta-cyclodextrin showed a positive effect on the aggregation suppression of both proteins. The influence of different L-arginine, L-Arginine ethyl ester, methyl-beta-cyclodextrin and 2-hydroxypropyl-beta-cyclodextrin concentrations on the renaturation yield of both proteins was investigated. Moreover, those additives did not only suppress the folding-related, but also temperature-related aggregates formation of both proteins. Since the solution additives effect on each protein was different, the main goal of the thesis was to elucidate the mechanisms by which L-arginine and cyclodextrins affect the aggregate formation of two model proteins, mink and porcine growth hormones, in two model systems: refolding from IBs and thermal denaturation. |