Recombinant Protein Production in <i>E. coli</i> Using the phoA Expression System

Abstract

Auto-inducible promoter systems have been reported to increase soluble product formation in the periplasm of E. coli compared to inducer-dependent systems. In this study, we investigated the phosphate (PO4)-sensitive phoA expression system (pAT) for the production of a recombinant model antigen-binding fragment (Fab) in the periplasm of E. coli in detail. We explored the impact of non-limiting and limiting PO4 conditions on strain physiology as well as Fab productivity. We compared different methods for extracellular PO4 detection, identifying automated colorimetric measurement to be most suitable for at-line PO4 monitoring. We showed that PO4 limitation boosts phoA-based gene expression, however, the product was already formed at non-limiting PO4 conditions, indicating leaky expression. Furthermore, cultivation under PO4 limitation caused physiological changes ultimately resulting in a metabolic breakdown at PO4 starvation. Finally, we give recommendations for process optimization with the phoA expression system. In summary, our study provides very detailed information on the E. coli phoA expression system, thus extending the existing knowledge of this system, and underlines its high potential for the successful production of periplasmic products in E. coli.

Keywords

• <i>E. coli</i>
• phoA promoter
• T7lac promoter
• pAT
• pET
• antibody fragment