EXPRESSION AND MUTAGENESIS OF RECOMBINANT HUMAN AND MURINE ERYTHROPOIETINS IN ESCHERICHIA-COLI

Expression of the polypeptide hormone erythropoietin (EPO) in Escheric hia coli by four bacterial expression vectors was examined. Complement ary DNAs encoding human and murine EPO were amplified by polymerase ch ain reaction (PCR) and cloned into the glutathione-S-transferase (GST) fusion vector, pGEX-2T. Human EPO DNA was also cloned into the vector s, pET14b, pIN III-Omp A2 and pT7/7. Expression of human and murine EP O was obtained using constructs based on pGEX-2T. For constructs based on the other vectors, expression of EPO was absent or occurred at low levels, despite attempts to optimise conditions. Human and murine EPO , expressed as fusion proteins with GST, were partially soluble and di splayed EPO bioactivity. Soluble GST-EPO fusion proteins were affinity purified on immobilised glutathione. Insoluble protein could also be purified by elution from gel slices following SDS-PAGE to yield either fusion protein or, after treatment with thrombin, unmodified EPO whic h was both soluble and bioactive. The pGEX expression system was evalu ated as a means of analysing the structure-function relationship of EP O by in vitro mutagenesis. Three human and three murine EPO mutants we re constructed and expressed as GST fusion proteins. Following purific ation, biological activity was evaluated using assays for bioactivity, immunoactivity and GST activity. The pGEX expression system complemen ts eukaryotic systems described previously for expression of EPO and s hould provide much useful information about the structure-function rel ationships of the hormone.