Many commonly used bacterial promoters employed for recombinant protein production (RPP) in Escherichia coli are capable of high-level protein expression. However, such promoter systems are often too strong, being ill suited for expressing proteins that are difficult to fold, targeted to the membrane or secreted out of the cytoplasm. To circumvent this problem, a suite of bacterial promoters has been constructed with a range of different promoter strengths, assigning them specific "promoter activity ratings" (PARs). Selecting three of these PAR promoters, with low, intermediate and high strengths, it is demonstrated that the expression of target proteins, such as green fluorescent protein (GFP), human growth hormone (hGH) and single chain variable region antibody fragments (scFvs), can be set to three levels when expressed in E. coli. It is shown that the PAR promoter system is extremely flexible, operating in a variety of E. coli strains and under various different culture regimes. Furthermore, due to its tight regulation, it is shown that this system can also express a toxic outer membrane protein, at levels which do not affect bacterial growth. Thus, the PAR promoter system can be used to tailor the expression levels of target proteins in E. coli and maximize RPP.
Bibliographical noteFunding Information:
J.H. was generously supported by an Industrial Biotechnology Catalyst award (Innovate UK, BBSRC, EPSRC) ( BB/M018261/1 ) to support the translation, development and commercialisation of innovative Industrial Biotechnology processes and by a BBSRC IAA Follow-on-Fund award ( BBSRC IAA BB/S506709/1 ). D.F.B was supported by BBSRC grants BB/M018261/1 and BB/R017689/1 . C.F. was supported by a studentship from the BBSRC Midlands Integrative Biosciences Training Programme .
- Escherichia coli/genetics
- Gene Expression Regulation, Bacterial
- Green Fluorescent Proteins/biosynthesis
- Human Growth Hormone/biosynthesis
- Promoter Regions, Genetic
- Recombinant Proteins/biosynthesis
- Single-Chain Antibodies/biosynthesis
- Recombinant protein production
- Lac promoter
- Escherichia coli
- Membrane proteins
- Transcription regulation