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

Abstract

Background: Production 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.

Results: The 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.

Conclusions: The 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.

Figure 1

Illustration showing how the different constructs in the study were generated based on pSB-M1b. The upper part shows how the alternative regulator/promoter systems were incorporated. pSB-M1b-1-17 contains a variant of the Pm core promoter termed ML1-17 (see text). The lower part shows the oriV/trfA region in pSB-M1b that was replaced with the pMB1 ori described in Table 1.

Figure 2

Maximum expression of three different genes placed under control of different regulator/promoter systems. Data represent relative expression levels under induced conditions where the activity of M1x (gene x under conrol of the Pm wildtype promoter, RK2 replicon) was set to 1.0. Expression was induced in a way that activity levels were maximized: 2 mM m-toluate for strains harboring XylS/Pm- based constructs, 1 mM IPTG for those with LacI/PT7lac, 0.2 mM IPTG for LacI/Ptrc and 0.015% L-arabinose for AraC/PBAD. The following E. coli strains were used as expression hosts. Panels A-C: ER2566. Panels D-F: DH10B. The naming code is the following: The capital letter represents the regulator/promoter system and the digit represents the origin of replication; for details see Table 1. The data presented are from independent biological replica.

Figure 3

Correlation between the accumulated transcript and protein produced after induction. The five proteins (Panels A-E) were expressed in E. coli ER2566 harboring pMB1-based plasmids. Five hours after induction, samples were collected for relative quantification real-time RT-PCR (qRT-PCR) and SDS-PAGE. Accumulated transcript data were correlated to the XylS/Pm system (M2x; gene x under conrol of the Pm wildtype promoter, pMB1 replicon). The total protein fractions were separated into the soluble supernatant and the insoluble pellet fraction after sonication and separated through SDS-PAGE followed by staining with Coomassie Brilliant blue. Further information about the naming system can be found in Table 1. Neg: Negative control.

Figure 4

Investigation of the tightness of different regulator/promoter systems in combination with the pMB1 replicon. Protein activity was determined in parallel with induced cultures at the time point corresponding to five hours after induction. The data presented are from independet biological replica. The following E. coli strains were used as expression hosts. Panels A-C: ER2566. Panels D-E: DH10B. The capital letters represent the regulator/ promoter systems according to Table 1. Uninduced expression of GFP in DH10B was very close to the detection limit in LB medium and was left out.

Figure 5

Theoretical analysis of the translational start site by calculating the translation initiation rate (TIR). The complete 5^′-UTR sequences in combination with the first 50 nucleotides of the respective genes (luc_S (Panel A), scFv173-2-5-phoA (B), gfpmut3 (C), GH1_S (D) and IL1RN_S (E)) were used as input sequences for the RBS calculator [54].

Figure 6

Analysis of the distribution of expression using flow cytometry. Strains were grown under standard conditions. At the time point of induction (t= 0 min) and at several points afterwards (t= 20–300 min), samples were collected, snap-frozen, and collectively analyzed with a flow cytometer. The spread is represented by the coefficient of variation (CV). Panels A-D: ER2566, Panel E: DH10B.