A comparative analysis of the properties of regulated promoter systems commonly used for recombinant gene expression in Escherichia coli

Abstract

BackgroundProduction of recombinant proteins in bacteria for academic and commercial purposes is a well established field; however the outcomes of process developments for specific proteins are still often unpredictable. One reason is the limited understanding of the performance of expression cassettes relative to each other due to different genetic contexts. Here we report the results of a systematic study aiming at exclusively comparing commonly used regulator/promoter systems by standardizing the designs of the replicon backbones.ResultsThe vectors used in this study are based on either the RK2- or the pMB1- origin of replication and contain the regulator/promoter regions of XylS/Pm (wild-type), XylS/Pm ML1-17 (a Pm variant), LacI/PT7lac, LacI/Ptrc and AraC/PBAD to control expression of different proteins with various origins. Generally and not unexpected high expression levels correlate with high replicon copy number and the LacI/PT7lac system generates more transcript than all the four other cassettes. However, this transcriptional feature does not always lead to a correspondingly more efficient protein production, particularly if protein functionality is considered. In most cases the XylS/Pm ML1-17 and LacI/PT7lac systems gave rise to the highest amounts of functional protein production, and the XylS/Pm ML1-17 is the most flexible in the sense that it does not require any specific features of the host. The AraC/PBAD system is very good with respect to tightness, and a commonly used bioinformatics prediction tool (RBS calculator) suggested that it has the most translation-efficient UTR. Expression was also studied by flow cytometry in individual cells, and the results indicate that cell to cell heterogeneity is very relevant for understanding protein production at the population level.ConclusionsThe choice of expression system needs to be evaluated for each specific case, but we believe that the standardized vectors developed for this study can be used to more easily identify the nature of case-specific bottlenecks. By then taking into account the relevant characteristics of each expression cassette it will be easier to make the best choice with respect to the goal of achieving high levels of protein expression in functional or non-functional form.

Production of prone‐to‐aggregate proteins

“…However, this strategy is sometimes difficult to scale up due to variation in media formulations. In conclusion, the ability to control expression levels is a key element in the choice of bacterial expression vectors [9,10].…”

Expression, characterization and mutagenesis of an FAD-dependent glucose dehydrogenase from Aspergillus terreus

“…Extensive work has been carried out to improve E. coli as a host for recombinant protein expression [21,31]. It is well known that promoter plays an important role in protein heterologous production, and is particularly important for producing functional protein [32]. In most conditions, the strong T7 promoter results in high-level expression, although many proteins were expressed by using this system, including FAD-GDH, they are not correctly folded and end up as inclusion bodies.…”

Designer Microorganisms for Optimized Redox Cascade Reactions – Challenges and Future Perspectives

“…[83] Recently, Balzer et al re-examined commonly used promoter systems in E. coli (XylS/P m , LacI/P T7lac , LacI/P tac ,a nd AraC/P BAD )a nd conducted comparative expression studies. [84] LacI/P T7lac turned out as highly advantageousa tatranscriptional level. Contradictory,o nt he level of translational, transcriptsw ere often translated into vast amountso fi nactive (insoluble) proteins.…”

“…Contradictory,o nt he level of translational, transcriptsw ere often translated into vast amountso fi nactive (insoluble) proteins. [84] This is long known for the popular pET system which also utilizes the LacI/P T7lac promoter. [85] Gene expression is induced by IPTG, al actose analogue that is not readily metabolized granting prolonged high expression levels.P romoters for metabolice ngineering must show tight control to avoid undesiredm etabolic load from leaky expression which is the case with the T7 expression system.…”