A comparative analysis of the properties of regulated promoter systems commonly used for recombinant gene expression in Escherichia coli
© Balzer et al.; licensee BioMed Central Ltd. 2013
Received: 9 November 2012
Accepted: 1 March 2013
Published: 18 March 2013
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.
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/P T7lac , LacI/P trc and AraC/P BAD 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/P T7lac 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/P T7lac 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/P BAD 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.
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.
KeywordsRecombinant expression Regulator/promoter systems XylS/Pm XylS/Pm ML1-17 LacI/P T7lac LacI/P trc AraC/P BAD Systematic comparison
Parameters affecting recombinant protein expression in Escherichia coli have been studied extensively and numerous methods aiming at improving protein yields have been reported, usually involving genetic manipulations and/or production process optimization [1–4]. However, in spite of the large number of potentially useful approaches available there is still no guarantee that a satisfactory result will be obtained in each specific case, and trial and error is therefore currently an integrated part of development of new protein production processes. The work involved in this can become very laborious since many parameters such as choice of strains, vector construct designs, growth media and cultivation conditions can potentially have a big and unpredictable effect on the process. Steadily more promoter systems for regulated protein expression in E. coli ( and references therein, [2–6]) are being developed, increasing the complexity. The studies of those novel expression systems were commonly based on experiments involving vectors with different backbones [2, 4, 7, 8]; typically commercially available and commonly used vectors from the pET , pTrc  or pBAD  series. More theoretical approaches have also been used [6, 12]. However, expression is influenced by many parameters even within vectors, like the presence or absence of sequences of the 5′ coding region encoding N-terminal fusion partners (His6 tag , N-terminal signal peptides , and others), different origins of replication [15–17], different terminators  or selection markers. Penicillins for example are very frequently used for selection in spite of their known rapid degradation due to secreted β-lactamase . A first step towards a more systematic, backbone-independent approach is described in a study performed by Tegel et al.  in which expression from three different IPTG-inducible promoters (P T7lac , P trc , P lac ) is compared. These are all based on the negative regulator LacI, while positively regulated promoters such as P BAD and Pm have not been used in such comparative studies. The regulators of these two promoters (AraC and XylS, respectively) are both members of the same family of transcriptional activators . The AraC/P BAD system is quite extensively used and its characteristics have been reviewed . The XylS/Pm system was included because it has several beneficial traits for protein expression in general (reviewed by Brautaset et al. ), and in combination with RK2 minimal replicons it has been demonstrated to be capable of expressing proteins at industrial levels in high cell density cultivations [14, 22], We have used this system extensively in our laboratory as a model for studies of recombinant gene expression. Particular advantages of this system are that the levels of expression can be fine-tuned by various means [23–25], that it is not host-dependent in contrast to most other systems and that the inducer is cheap. Furthermore, expression from the native system could be greatly improved by generating variants of the regulator protein XylS , the DNA region corresponding to the Pm promoter region  as well as the region corresponding to the Pm 5′-untranslated region (5′-UTR) .
In this report we describe a systematic comparison of both positively and negatively regulated expression systems. Being aware of the influence of the 5′ end of the coding region on expression [29, 30], we intentionally chose to use model genes with native 5′ ends as opposed to commonly used regions encoding N-terminal detection tags or solubility-enhancing fusion partners. The expression analyses were carried out at both the transcript and the protein level (activity assays and total protein), and we also included a flow cytometry based analysis of expression in individual cells. All comparisons were performed using identical vector backbones, a procedure we believe can be used generally as a diagnostic tool to identify bottlenecks in recombinant protein production processes.
Results and discussion
Construction of a set of plasmids specifically designed for comparative studies of commonly used expression systems in E. coli
Plasmids used in this study a
m-toluate- inducible Pm, xylS activator gene, RK2 replicon, bla reporter, Kanr
IPTG-inducible P T7lac , lacI repressor gene, Ampr
L-arabinose- inducible P BAD , araC activator gene, Ampr
IPTG- inducible P trc promoter, lacI repressor gene, Ampr
pIB11  with luc S under control of xylS/Pm, Kanr
pBAD24 with gfpmut3 insert, Ampr
pHOG plasmid with scFv173-2-5-phoA fusion gene insert, provided by Affitech AS, Oslo, Ampr
pMA vector (GeneArt®, Invitrogen) with GH1 S insert, provided by Vectron Biosolutions AS, Trondheim, Ampr
pMA vector (GeneArt®, Invitrogen) with IL1RN S insert, provided by Vectron Biosolutions AS, Trondheim, Ampr
pSB-M1b variants with combinations of different features:
P… regulator/promoter system
M… xylS/P m
M-1-17… xylS/P m variant ML1-17
E… lacI/P T7lac (from pE T)
T… lacI q /P trc (from pT rc)
B… araC/P BAD (pB AD)
0… origin of replication
1… RK2 replicon
2… pMB1 replicon
x… reporter gene
l… luc S
h… GH1 S
r… IL1RN S
m-toluate- inducible P m , xylS activator gene, pMB1 ori, luc S reporter, Kanr
Properties of the proteins selected as expression reporters
reporter protein, ~ 60.8 kDa, cytoplasmic localization, generally low expression, rather easy to detect, very sensitive detection via bioluminescence assay
industrially relevant protein, ~77.2 kDa, fusion protein, disulfide bonds, translocated to the periplasm, detectable through APa fusion, AP needs to be translocated to be active 
reporter protein, ~ 26.9 kDa, cytoplasmic localization, stable, known to be produced virtually only in its soluble form , very easy to detect by direct fluorometry
Due to the nature of the expression systems it was necessary to use two different E. coli strains as hosts. Strain ER2566 was chosen to compare expression from LacI/P T7lac with XylS/Pm because it carries a chromosomal copy of the T7 polymerase integrated into the lac operon (NEB). Since the LacI/P trc system is also induced by IPTG, it was decided to study expression in the same host under the assumption that the expression of T7 polymerase does not affect expression from LacI/P trc due to the specificity of this polymerase for its cognate promoter . Expression from XylS/Pm compared to AraC/P BAD was performed in E. coli DH10B which is unable to catabolize L-arabinose, the inducer of the AraC/P BAD system.
Protein production levels are generally stimulated by increased gene dosage, but none of the tested cassettes are superior for all genes
The LacI/P T7lac system is unique by its generation of large amounts of transcript and insoluble protein
For GFP and HGH (Panels C and D) production of soluble protein was very effective in both XylS/Pm ML1-17 and LacI/P T7lac , and the final outcome at the protein level was more similar for these proteins than for luciferase. Generally, LacI/P T7lac had an apparent advantage by its performance at the transcriptional level, but this potential was often not reflected at the translational level, such that the system often produced a vast amount of transcripts that were either translated into inactive protein or were not translated at all. Note also that the amounts of protein and transcript correlated well for XylS/Pm and XylS/Pm ML1-17 (except for scFv173-2-5-AP, Panel B), probably mainly because the amounts of transcript were generally much lower than for LacI/P T7lac and therefore did not overload the translational machinery. It is also interesting to note that, in terms of both active and total protein produced, XylS/Pm ML1-17 and LacI/P T7lac generally performed best. For scFv173-2-5-AP (Figure 3, Panel B) a more complex picture was observed, but this could be mainly related to the effects of toxic protein production on host growth or variability among the systems in the kinetics of induction .
Uninduced expression levels are highest for LacI/P trc and lowest for AraC/P BAD
Generally, LacI/P trc tended to be the leakiest system producing 3.8 to 8.2 times more active protein than XylS/Pm under uninduced conditions. Similarly, XylS/Pm ML1-17 displayed 2.8- to 5.8-fold higher background expression than the wild-type system. AraC/P BAD appeared to be, as expected, the tightest system giving rise to 0.1 and 0.4 times the background level for luciferase and scFv173-2-5-AP, respectively. LacI/P T7lac was also quite tightly regulated although it generated the highest background expression for GFP (Figure 4, Panel C).
The ratio between the induced and the uninduced expression levels was protein dependent with relatively small induction windows for svFv173-2-5-AP (1.2-25) and large for luciferase (60–3,000). In strain ER2566, XylS/Pm and LacI/P T7lac displayed the highest induction windows, while LacI/P trc was by far the least inducible system (0.1-0.2 times compared to XylS/Pm). In DH10B, induction ratios for AraC/P BAD were 1.3-27 times higher than the ratios of XylS/Pm and XylS/Pm ML1-17. These results are consistent with a previous report documenting that the induction ratio in the AraC/P BAD system can reach up to 1,200-fold when functionally compared for the phoA reporter gene . As for XylS/Pm[24, 25], the induction level can also be modulated over a wide concentration range by varying the inducer concentration. In addition, uninduced levels can be even further reduced by the presence of glucose, which represses the expression in this system . The main disadvantage of the AraC/P BAD system is that the inducer can be metabolized in most strains of E. coli.
The predicted translational efficiencies of the ribosomal binding sites vary over a wide range
To correlate the calculated TIR values with our experimental data is not straight forward because the total protein levels are obviously also dependent on the efficiencies of the promoter sequences, which are not a part of the calculation of the TIR values. However, by comparing both transcript and protein amounts available from the data presented in Figure 3 such effects can at least partly be taken into account. The amounts of accumulated transcripts derived from LacI/P T7lac were generally highest and combined with a predicted more efficient TIR one might expect that this system would come out best at the protein level in all cases. However, this prediction was only in agreement with the luciferase data, and with the ScFv-173-2-5-AP and IL-1RA data to a more limited extent. In contrast, for GFP and HGH the experimental data did not support the prediction. It should also be remembered that efficient translation in itself may contribute to more accumulated transcript due to translation-mediated transcript stabilization [55, 56]. For XylS/Pm ML1-17 there appeared to be more protein per transcript compared to LacI/P T7lac and the total amounts of protein were at least equally good for this system, presumably indicating a better balance between the capacities of the transcriptional and translational systems. For LacI/P trc the calculator correctly predicted a very poor expression of HGH.
In general, it is possible to some extent to use the RBS calculator to predict which regulator/promoter system would produce most protein. However, RBS function is just one among several parameters that affect the final protein production level. We have analyzed the previously reported very efficient UTR variants obtained by screening . Despite the great stimulatory effect of these screened UTRs on protein expression (up to 20-fold), the calculator only predicted minor improvements relative to the wild-type sequence (between 1.5 and 3.6 times for the best variants).
Flow cytometry analysis of GFP expression in individual cells revealed significant differences among the various regulator/promoter systems
Analyses of recombinant protein expression are mostly carried out at the level of cell populations, potentially masking significant differences in the level of expressed proteins between individual cells, which are known to occur [57, 58]. If such heterogeneity exists it may represent another possibility for system improvement, e.g. by finding ways to reduce the fraction of cells with low expression level. This is also relevant in metabolic engineering projects involving metabolite flux control in biochemical pathways .
The LacI/P trc system (Figure 6, Panel D) is characterized by a very even signal distribution throughout the entire induction period. Interestingly, the mean fluorescence remained constant already two hours after induction, possibly a consequence of a very fast activation of transcription after inducer addition in this system.
The AraC/P BAD system, displayed a similar behaviour as XylS/Pm meaning that it takes an extended time until all cells are induced as reflected by a tail of the distribution towards low fluorescence values (Figure 6, Panel E). One hour after induction, the distribution fell into a single, rather narrow peak that was shifted towards higher fluorescence values over time.
The outcomes of the flow cytometry experiments showed that there is a quite big variation in GFP expression level among individual cells. By better understanding the factors controlling this variability it may become possible to improve expression at the population level. This conclusion is supported by the observation mutations in the Pm promoter region lead to more homogeneity.
Summary of the findings derived from the comparative expression study
XylS/Pm and Pm ML1-17
Pm promoter (native or variant)
trp/lac hybrid promoter
P BAD promoter
CAP binding site
CAP binding site
strain supplying T7 polymerase
araBADC-/ araEFGH+ strain
Range of inducer
0.001 - 2.0 mM
0.05 - 2.0 mM
0.05 - 2.0 mM
0.001% - 1%
low - high
intermediate - high
low - intermediate
intermediate - high
low - high
low - high
low - intermediate
below detection - intermediate
weak - intermediate
intermediate - strong
weak - intermediate
high level expression
high level expression
(high level expression)c
high level expression
expression of toxic proteins
(expression of toxic proteins)c
expression of toxic proteins
Strains, standard DNA manipulations and growth conditions
E. coli DH5α (Bethesda Research Laboratories) was used for plasmid propagation during cloning steps. Recombinant DH5α strains were grown at 37°C in liquid Luria Bertani (LB) broth or on solid LB plates with appropriate antibiotics (kanamycin 50 μg/mL; ampicillin 200 μg/mL). E. coli ER2566 (New England Biolabs, NEB) and E. coli DH10B (Invitrogen) served as expression hosts during the comparative studies. In comparison to the commonly used strain E. coli BL21(DE3), the former strain offers higher transformation efficiency for toxic clones and less background expression (NEB). All DNA manipulations were carried out according to standard procedures  or according to manufacturers’ instructions. PCR was performed using the Expand High Fidelity PCR systems kit (Roche), and essential regions in PCR products were verified by sequencing. Functionality of the regulator/promoter systems was confirmed using bla as reporter gene determining the levels of resistance to ampicillin as described previously .
Oligonucleotides used in this study
Sequence (5′→ 3′)
a) PCR primers
b) qRT-PCR primers
Construction of pSB-M2b: The region of pBAD_gIII_calmodulin containing the origin of replication from pMB1 was PCR amplified using primer pair Pwitw6_badF and Pwitw6_badR. In parallel, pair Pwitw4_AscI and Pwitw5_SpeI was used to amplify pSB-M1b  excluding the RK2 ori (trfA coding region and the oriV origin of replication). After digestion with AscI and SpeI of both the amplified pMB1 ori and the pSB-M1b -resulting PCR product, the two fragments were ligated to each other resulting in plasmid pSB-M2b. The difference between copy-numbers of RK2- and pMB1-based plasmids was confirmed by agarose gel electrophoresis. Construction of pSB-P0b introducing different regulator/promoter systems: Three different regulator/promoter systems were chosen to substitute the region spanning xylS/Pm in pSB-M1b and pSB-M2b. The lacI/P T7lac region was amplified from pET16b using ET_AgeI_fwd and ET_NdeI_rev and inserted into the two depicted backbones using NdeI and AgeI, generating pSB-E1b and pSB-E2b. The lacI q /P trc region was amplified from pTrc99A using TRC_AgeI_fwd1and TRC_NdeI_rev1 prior to insertion into pSB-M1b and pSB-M2b using AgeI and NdeI, generating pSB-T1b and pSB-T2b. Finally, the PCR product covering the araC/P BAD region from pBAD/gIII_calmodulin generated with the primers BAD_BbsI_fwd and BAD_NdeI_rev was inserted into the above mentioned backbones using BbsI and NdeI, creating pSB-B1b and pSB-B2b. In order to insert the Pm variant ML1-17 , pSB-M1b and pSB-M2b were digested with XbaI and PciI removing the Pm core promoter region which was replaced by two annealed oligonucleotides that constitute the double-stranded Pm ML1-17 fragment with XbaI and PciI compatible ends, creating pSB-M1b-1-17 and pSB-M2b-1-17. Introduction of other genes of interest: All pSB-P0b variants, except for pSB-B2b, were digested with NdeI and BamHI to excise the bla gene and to insert the lucS gene from pIB11-lucS instead, generating pSB-P0l variants. pSB-B2b and pSB-M1l were digested with NdeI and KpnI. The resulting DNA fragment corresponding to the pSB-B2 backbone and the lucS gene were ligated to each other to generate pSB-B2l. The scFv173-2-5-phoA gene was PCR cloned from pHOG-173-2-5-AP with primer pair pelB_fwd and APhis_rev2. The enzyme combination NdeI and BamHI was used to replace the bla gene from pSB-M1b with the digested scFv173-2-5-phoA PCR product resulting in pSB-M1s. From there on NdeI and BamHI were used to generate all pSB-P0s variants, except for pSB-B2s. This construct was generated by digesting pSB-B2b and pSB-B1s with BamHI and ligating the pSB-B2 backbone with the scFv173-2-5-phoA BamHI digested insert from pSB-B1s. gfpmut3 originating from pBAD24-GFP was inserted into the pSB-P0b variants using NdeI and BamHI with the exception of pSB-B2b. Instead, BamHI was used to excise the gene from pSB-B1g and to place it into pSB-B2 backbone (originating from pSB-B2l) to generate pSB-B2g. Genes GH1S and IL1RNS were excised from pMA-GH and pMA-T-IL-1RA with NdeI and BamHI and transferred to the pSB-P0b variants with the Pm, Pm ML1-17, P T7lac and P trc promoter using the same enzymes, resulting in pSB-P0h and pSB-P0r variants.
Growth conditions for comparative expression studies
The general cultivation protocol was based on recommendations published by the European Molecular Biology Laboratory (EMBL) . For E. coli cultivations LB medium was chosen because it is widely used among molecular biologists and at the same time it was avoided to use media with glucose as a carbon source due to the influence of glucose on background expression from P T7lac and P BAD through catabolite repression . A growth temperature of 30°C was applied for slowing down the growth rate of E. coli, as this generally leads to more soluble protein . Initially the kinetics of protein accumulation was studied for all expression cassettes, using GFP (fluorescence) and luciferase (activity) as the main models.The inducer concentrations and culture harvesting times post induction were varied and we found that five hours induction was sufficient to reach a plateau of accumulated protein per OD unit of cells. For GFP the accumulation rate was nearly constant (slightly lower from 3–5 hours) over this time-period. For most of the proteins it was complicated to follow the kinetics accurately since there was no quantitative method for measurement available, and in case of luciferase activity measurements may not necessarily correlate exactly with the accumulation kinetics of the insoluble fraction.
Recombinant E. coli ER2566 and DH10B strains were grown in 2 ml LB supplemented with 50 μg/ml kanamycin at 30°C over-night. Then 15 ml of LB with kanamycin in shake flasks were inoculated with the overnight culture to an initial OD600 of 0.05. Following incubation at 200 rpm and 30°C expression was induced at OD600= 0.5-0.6 as follows: 2 mM m-toluate for strains harboring Pm- based constructs, 1 mM IPTG for those with P T7lac , 0.2 mM IPTG for P trc and 0.015% L-arabinose for P BAD . Growth was continued for 5 more hours at 30°C.
Transcript analysis by qRT-PCR
At harvest, 0.5 ml of culture was stabilized with RNA protect (Qiagen) prior to freezing. The subsequent total RNA isolation, cDNA synthesis and relative transcript quantification by qRT-PCR was performed as described previously . Primer pairs used during amplification are listed in Table 4. Transcript generated from the 16S rRNA gene was used for normalization.
Activity measurements of the different reporters
The luciferase assay was performed using the Luciferase assay System (Promega). At harvest, the cell culture was normalized to an OD600 of 0.5. 90 μL of this mixture was supplemented with 10 μL of K2HPO4, pH 7.8, 20 mM EDTA prior to lysis with the Luciferase Cell Culture Lysis Reagent (CCLR, Promega). The remaining steps of the protocol were carried out according to the manufacturer’s instructions except that the luciferase activities were determined from 10 μL lysed culture mixed with 50 μL of substrate. The alkaline phosphatase assay was performed as described previously . Fluorescence measurements of strains expressing GFP were performed with the FLUOstar Omega instrument (BMG Labtech) together with the corresponding Omega Software. Fluorescence intensity was determined directly from the cultures using an appropriate filter set (excitation: 485 nm; emission: 520 nm). Values were normalized against the optical density. Data were acquired from three biological and thereof three technical replica.
Protein analysis by SDS-PAGE
For SDS-PAGE analysis 50 ml culture volume was used. Because of impaired growth of recombinant strains expressing scFv173-2-5-AP, 3xLB was used to get sufficient cell mass for analysis. The general growth conditions were as described above for the comparative expression studies. At harvest, bacterial pellets were washed with 0.9% NaCl and 100 mg pellet (wet weight) was frozen until further processing. Pellets were resuspended in lysis buffer (50 mM Tris–HCl, pH 8.0, 1 mM EDTA, 100 mM NaCl, 8 mM MgCl2). The solution was sonicated using a Branson Sonifier DSM tip (sonication for 3.5 minutes on ice, duty cycle 35% and output control 3.0). Soluble and insoluble fractions were separated by centrifugation and treated with 62.5 U/ml Benzonase nuclease (Merck). Protein gels were run under denaturing conditions using ClearPAGE 10% gels and ClearPAGE SDS-R Run buffer (C.B.S. Scientific) followed by staining with Coomassie Brilliant blue R-250 (Merck).
Cultures were grown essentially as decribed for SDS-PAGE analysis. At various time points after induction, 1 ml of culture was collected, supplemented with glycerol to 10% and snap-frozen in liquid nitrogen until further analysis. For single-cell fluorescence measurements, samples were thawed on ice and diluted in PBS. Flow cytometry was performed using the CyFlow® Space flow cytometer (Partec) equipped with a 488 nm blue solid state laser (200 mW) and a 536/ 40 nm band pass filter. For each sample, 150,000 events were collected at a rate between 800 and 2,000 events per second. Data were analysed with the Windows™ XP FloMax(R) software (Quantum Analysis). The mean and spread (coefficient of variation (CV)) of the distributions were calculated over all collected values after gating.
Relative quantification real-time RT-PCR
Single-chain antibody fragment 173-2-5 alkaline phosphatase fusion protein
Green fluorescent protein
Human growth hormone
Human interleukin 1 receptor antagonist
European Molecular Biology Laboratory
Sodium dodecyl sulfate- polyacrylamide gel electrophoresis
Ribosome binding site
Translation initiation rate
Coefficient of variation
New England Biolabs
We a very grateful to Laila Berg for the gift of pIB11-lucS, Affitech, AS, Oslo for providing pHOG-173-2-5-AP and Vectron Biosolutions AS, Trondheim, for the gift of pMA-GH and pMA-T-IL-1RA. We thank Andrea Ebert for assisting with the collection of the flow cytometry data. This work was funded by a stipend that SB obtained from the Faculty of Natural Sciences and Technology, NTNU, Trondheim.
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