In this thesis, production of the soluble protein tissue-type plasminogen activator variant (rPA) in the periplasm and culture supernatant with the assistance of thiol oxidase DsbA as a coexpressed helper protein in shake flask and fed-batch cultivation was investigated. The influences of coexpression of DsbA, different additives (L-arginine, redox system, glycine and Triton X-100) and fermentation conditions on the localization of rPA in the periplasm and culture supernatant were investigated. Besides that, a new technique Dynamic Light Scattering (DLS) using the Zetasizer 3000 (Malvern, UK) was applied to determine the impact of production conditions on the size of inclusion bodies (IBs) of α-glucosidase in cytolasm of E.coli and on the protein solubility. Size distributions of IBs of α-glucosidase under various cultivation conditions such as: different media, temperature, batch and fed-batch cultivation at different temperature were analysed by DLS. By overproduction of several molecular chaperones e.g. DnaK and ClpB in E. coli, effects of those chaperones on aggregated protein formation, resolubilization of α-glucosidase IBs and the relation of α-glucosidase activity to the size of the IBs were also studied. |