Purification of therapeutic proteins mediated by in vivo polyester immobilized sortase
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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 (TNFα) and human interferon alpha 2b (IFNα2b) by Escherichia coli.
Hybrid genes encoding PhaC-Sortase-TNFα or PhaC-Sortase-IFNα2b fusions (with a LPETG recognition signal immediately before TNFα or IFNα2b), mediated intracellular production of polyester (polyhydroxyalkanoate, PHA) beads in Escherichia coli. Upon isolation of respective PHA beads, pure soluble TNFα or IFNα2b was released by activating sortase via addition of CaCl2 and triglycine. TNFα and IFNα2b 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.
KeywordsProtein purification Polyhydroxyalkanoate Polyhydroxyalkanoate synthase Sortase Self-cleavage Therapeutic proteins
This work was funded by Griffith University (Australia) and Massey University (New Zealand). We specially thank Iain D. Hay for preparation of plasmid pET14-PhaC-SrtA-TNFα and pET14-PhaC-SrtA-IFNα2b. We thank Trevor Loo for carrying out the LC–MS/MS analysis on all the pre-digested protein samples.
Supplementary Table 1—Bacterial strains and plasmids used in this study.
Supplementary Fig. 1—LC–MS/MS analysis result for the purified therapeutic proteins.
Supplementary Fig. 2—LC–MS/MS analysis result for the co-purified carrying-over proteins.
Compliance with ethical standards
BHAR is co-founder, shareholder and Chief Technology Officer of Polybatics Ltd that commercializes products based on the PHA bead technology.