PARAMETERS INFLUENCING THE EXPRESSION OF MATURE GLIAL-CELL-LINE-DERIVED NEUROTROPHIC FACTOR IN ESCHERICHIA-COLI

Factors governing expression in Escherichia coil, namely promoters, fu sion partners, targeting signals, host strains, growth temperature of cultures and inducer concentrations, were investigated to elucidate th eir influence on the accumulation of mature glial-cell line-derived ne urotrophic factor (GDNF), The present study provided evidence indicati ng that translational and/or post-translational events were more impor tant in determining overall accumulation of the target protein than wa s transcription, Under the control of the strong inducible tac or T7 p romoter, no direct correlation between transcript abundance and final yield of recombinant protein was observed. GDNF was also recalcitrant to being produced in a soluble form in E. coli. Direct expression resu lted in the exclusive localization of GDNF in inclusion bodies, regard less of whether the protein was produced in the cytoplasm or targeted to the periplasm. The fusion approach was found to be the most efficie nt method, as it resulted not only in the highest level of GDNF produc ed, albeit primarily in inclusion bodies, but also in the accumulation of small amounts of soluble proteins, Using different host strains, l ow inducer concentration or sub-optimal growth temperature did not res ult in any detectable shift towards solubility. Persistent localizatio n in inclusion bodies, low levels of expression as a native protein an d in vivo proteolysis of soluble fusion forms appeared to be influence d by structural features located at the hi-terminus of GDNF. Deletion of this region was found to result in substantial alleviation of these problems.