To overcome laborious and costly procedures often associated with therapeutic protein production and purification, in vivo polyester immobilized sortase is explored for the production of human tumor necrosis factor alpha (TNF alpha) and human interferon alpha 2b (IFN alpha 2b) by Escherichia coli. Hybrid genes encoding PhaC-Sortase-TNF alpha or PhaC-Sortase-IFN alpha 2b fusions (with a LPETG recognition signal immediately before TNF alpha or IFN alpha 2b), mediated intracellular production of polyester (polyhydroxyalkanoate, PHA) beads in Escherichia coli. Upon isolation of respective PHA beads, pure soluble TNF alpha or IFN alpha 2b was released by activating sortase via addition of CaCl2 and triglycine. TNF alpha and IFN alpha 2b each were recognized by corresponding conformational antibodies in an ELISA assay. In vivo polyester immobilized sortase could be exploited for production and purification of high-value therapeutic proteins without laborious and costly downstream processing.