Use of glucose to control basal expression in the pET...
Transcript of Use of glucose to control basal expression in the pET...
Many popular bacterial ex-pression systems, includingthe pET system, containcomponents of the lac
operon. For example, in the pET systemtranscription of the target gene is controlledby a bacteriophage T7 promoter, and theproduction of the T7 RNA polymerase inexpression hosts (λDE3 lysogens) is regu-lated by a lac promoter derivative, the E.coli L8-UV5 lac promoter (see Figure 1).
Negative regulation by repressor
The wild type lac operon has two dis-tinct mechanisms of regulation; one is nega-tive (decreases transcription), and the otheris positive (stimulates transcription).Negative regulation is mediated by the lacrepressor. Transcription initiation beginswith the binding of E. coli RNA polymeraseto the promoter; however, the successfultransition from transcription initiation totranscription elongation can be influencedby downstream elements. Between the pro-moter and the coding regions in the operonis the lac operator, which is a specific DNAsequence to which lac repressor binds. Thebinding of repressor to the operator greatlydecreases the frequency of successful tran-scription elongation events by the RNApolymerase. Inducers of the lac operon (e.g.,IPTG) permit transcription because theybind to the lac repressor and substantiallydecrease its binding affinity to the lacoperator.
Positive regulation by CAP + cAMPand the glucose effect
It would seem that there should be littleto no expression in cells in the absence ofinducer and expression should proceedwhen an inducer is added. However, effi-cient transcription initiation also requiresthe presence of cyclic AMP (cAMP) andcyclic AMP receptor protein, called CRP orCAP. The CAP/cAMP complex binds justupstream of the lac promoter and directlystimulates transcription by RNA poly-merase. Because the binding of CAP toDNA requires cAMP, induction of tran-scription depends on the level of cAMP in
the cell. cAMP levels are strongly influencedby the carbon source present in themedium. In the presence of glucose (aneasily metabolized monosaccharide), cAMPlevels are low, so transcription from the lacpromoter is low. This phenomenon is calledthe glucose effect or catabolite repressionand is shared by a number of E. coli oper-ons. When glucose is absent and the cell isforced to use an alternative carbon source,such as glycerol, cAMP levels rise. The re-sulting formation of the CAP/cAMP com-plex stimulates transcription from the lacpromoter. Therefore, full induction of thelac operon is achieved only in the presenceof both inducer and elevated cAMP levels.
elements and other transcriptionalcontrols in the pET System
The lambda DE3 prophage encodingT7 RNA polymerase in pET expressionhosts carries the L8-UV5 promoter, whichhas three point mutations that distinguish itfrom the wild type lac promoter (Figure 1).Two point mutations in the –10 region in-crease promoter strength and decrease itsdependence on CAP/cAMP stimulation forfull activation. The third-point mutation islocated in the CAP/cAMP binding site anddecreases the affinity for CAP/cAMP. Thismutation reduces, but does not eliminate,sensitivity to catabolite repression. The neteffect of the three-point mutations is thecreation of a stronger promoter that is less
sensitive to the glucose effect. This allowsstrong IPTG induction of T7 RNA poly-merase expression even in the presence ofglucose.
Although the lac and L8-UV5 promot-ers are well repressed in the absence of in-ducer, both exhibit detectable basal activity.In the case of λDE3 lysogens, basal expres-sion of even a small amount of T7 RNApolymerase can lead to problems if thetarget gene in the pET vector produces aprotein toxic to the host cell. Therefore, ad-ditional levels of control are built into thepET vectors and hosts. Vectors with a“T7lac” promoter have a T7 promoter fol-lowed by a lac operator sequence. Theoperator in these plasmids provides a placefor lac repressor to bind, reducing transcrip-tion by any T7 RNA polymerase that maybe expressed in the absence of inducer.Another level of control is provided in ex-pression hosts containing the pLysS plas-mid, which expresses T7 lysozyme, a pro-tein that binds to and inhibits T7 RNApolymerase. The need for these additionalsources of regulation depends on the targetprotein being expressed; the more damagingthe protein is to bacterial cells, the moreregulation is required.
As first described by Grossman et al. (1),yet another level of regulation can be em-ployed with the pET System by exploitingthe glucose effect described above, i.e.,
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ARTICLE
Use of glucose to control basal expression in the pET SystemRobert Novy and Barbara Morris—Novagen, Inc.
—35 —10
lacI lacO2
RBS RBS
cAMP/CAPbinding site
L8-UV5 lac promoter
TTTACA TATAAT lacZ T7 gene 1
T7 RNA polymeraseTranscription start
CAP/cAMP
repressor
lacO1lacO3
E. coli RNA polymerase
Figure 1. Transcriptional control of T7 gene 1 in λDE3 lysogensTranscription of T7 gene 1 (encoding T7 RNA polymerase) in pET System expression hosts (λDE3 lysogens) is controlled by the L8-UV5lac promoter. T7 gene 1 is transcribed as the second gene in a bicistronic mRNA (the first gene contains an N-terminal fragment of lacZthat includes the α-peptide coding region). Positions of the three mutations of the wild type lac promoter region are indicated by coloredcircles. The lac repressor (lacI gene product) binds to lacO1, and then interacts with pseudo-operators lacO2 and lacO3 to prevent transcrip-tion by E. coli RNA polymerase. The inducer IPTG binds to the repressor, reducing its affinity for lacO1 and thus enabling transcription tooccur. When cAMP levels are sufficiently high (e.g., in the absence of glucose) the CAP/cAMP complex is formed and binds immediatelyupstream from the promoter to fully stimulate transcription. In the presence of glucose, CAP/cAMP is not formed and transcription is de-creased. This is called the glucose effect, or catabolite repression.
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supplementing standard media such as LBwith glucose to keep cAMP levels low.Apparently, although the L8-UV5 promoteris less dependent on CAP/cAMP stimula-tion than the wild type lac promoter, inpractice there is still a significant reductionin basal transcription in the presence of glu-cose. This can be particularly important forpET vector expression when hosts that donot carry the pLysS plasmid are allowed togrow to stationary phase, where uninducedexpression is maximal (1, 4). Others havealso reported that supplementing LB mediawith glucose to a final concentration of0.5–1.0% prevents the increased basal activ-ity observed in cultures grown to stationaryphase (1, 2).
Example of the glucose effect on pETexpression
Figure 2 demonstrates the dramatic dif-ference that glucose can make when cul-tures are grown to stationary phase byovernight incubation at 37°C. pET-30 re-combinants expressing green fluorescentprotein (GFP) in Tuner™(DE3) andTuner(DE3)pLysS hosts were tested underdifferent growth conditions. Cultures in LBmedium lacking or containing 1% glucosewere grown to stationary phase (16 hours)or to log phase (OD600 = 0.6 and 1.0). Thelog phase cultures were then induced withIPTG for 3 hours at 37°C. Each conditionwas carried out in duplicate with two inde-pendent recombinants. Cells were harvested
at the end of the culture period and totalcell protein (TCP) samples were analyzedby SDS-polyacrylamide gel electrophoresis.
Figure 2, panel A shows the gel profilesof the overnight cultures grown withoutadded inducer. The overnight Tuner(DE3)cultures lacking glucose (lanes 1–2) exhib-ited easily detectable levels of target proteinproduction, whereas target protein was un-detectable in the same cultures supple-mented with 1% glucose (lanes 5–6). Incontrast, the Tuner(DE3)pLysS culturesgrown to stationary phase did not requireglucose to prevent uninduced expression(lanes 3–4 without glucose vs. lanes 7–8
with glucose).Figure 2, panel B demonstrates that glu-
cose addition did not interfere with IPTGinduction of the target protein. In fact,IPTG induction from the pLysS host ap-peared to be enhanced in the presence ofglucose. High induction was observed fromthe Tuner(DE3) cultures regardless of glu-cose addition (panel B, lanes 1–2 vs. lanes5–6). Much lower expression of the targetprotein was observed from the pLysS-basedhost grown in glucose (lanes 7–8), but itwas barely detectable in the host grown
without glucose (lanes 3–4).One possible explanation for the low
IPTG induction results observed in thepLysS host is that in the absence of glucosethe expression of T7 lysozyme from pLysSmay be substantially elevated. In uninducedpLysS host cultures, some transcription ofthe LysS gene is probably achieved via read-through transcription from the upstreamchloramphenicol acetyltransferase (CAT)promoter. In the absence of glucose, cAMPlevels would be expected to rise during thelater stages of the growth cycle. Because theCAT promoter is also stimulated by theCAP/cAMP complex (3), elevated tran-scription of the lysozyme gene from theCAT promoter would occur. In Figure 2, aunique protein band was observed betweenthe 15 and 25 kDa protein markers inpLysS cultures, which corresponds to thepredicted size of T7 lysozyme (17 kDa).This band was significantly more intense inpLysS cultures grown without glucose rela-tive to those grown in the presence of glu-cose. A sufficiently high level of T7lysozyme may saturate all of the availableT7 RNA polymerase and thereby blocktarget gene transcription. This may accountfor the variability that is sometimes ob-served when attempting to induce targetproteins in pLysS hosts.
Summary
In conclusion, supplementing culturemedia with glucose provides a simple, inex-
A. Cultures grown to stationary phase (16 h) B. Cultures grown to OD600 = 0.6–1 and induced 3 h with IPTG
M 1 2 3 4 5 6 7 8kDa
150 –
100 –75 –
50 –
35 –
25 –
15 –
M 1 2 3 4 5 6 7 8
Figure 2. Expression of GFP from a pET-30 construct under different conditionsTuner(DE3) and Tuner(DE3)pLysS hosts carrying a pET-30 Ek/LIC GFPuv recombinant were grown under various conditions and analyzed for target protein expression by SDS-PAGE of total cell extracts. In PanelA, 3-ml cultures were grown to stationary phase by overnight incubation (16 h) at 37°C with shaking at 300 rpm. In Panel B, 3-ml cultures were grown to an OD600 between 0.6 and 1.0 and then induced by the ad-dition of 1 mM IPTG for 3 h at 37°C with shaking at 300 rpm. For gel analysis, cells were harvested by centrifugation and the pellets resuspended in BugBuster™ HT Protein Extraction Reagent. After the additionof 4X SDS Sample Buffer, samples corresponding to equivalent numbers of cells (based on harvest OD600) were loaded on a 10–20% gradient gel. In both panels, lanes 1–4 represent cultures grown in standardLB broth and lanes 5–8 represent cultures grown in LB broth supplemented with 1% glucose. The respective hosts are indicated. Pairs of lanes represent duplicate samples derived from independent clones.
M. Perfect Protein™ Markers (15–150 kDa)
Lane Host Medium1. Tuner(DE3) LB2. Tuner(DE3) LB3. Tuner(DE3)pLysS LB4. Tuner(DE3)pLysS LB5. Tuner(DE3) LB + glucose6. Tuner(DE3) LB + glucose7. Tuner(DE3)pLysS LB + glucose8. Tuner(DE3)pLysS LB + glucose
← Target protein
← T7 lysozyme
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Supplementing LB medium with 1% glucose prevents increased basal expression in DE3 lysogens grown
to stationary phase.
Sodium dodecyl sulfate polyacry-lamide gel electrophoresis (SDS-PAGE) is the most widely used ana-lytical method to resolve separate
components of a protein mixture. It isalmost obligatory to assess the purity of aprotein through an electrophoretic method.SDS-PAGE simultaneously exploits differ-ences in molecular size to resolve proteinsdiffering by as little as 1% in their elec-trophoretic mobility through the gel matrix(1). The technique is also a powerful tool forestimating the molecular weights of proteins(2, 3). The success of SDS-PAGE as an in-dispensable tool in protein analysis has beenattributed to three innovations that permit-ted the correlation of electrophoretic mobil-ity with a protein’s molecular mass (4). Firstwas the introduction of discontinuous buffersystems where the sample and gel runningbuffers differ in both composition, Tris-HCl/Tris-glycine, and pH, 6.8/8.3, respec-tively (5, 6). Discontinuous buffer systemsallow larger sample volumes to be loadedwhile maintaining good resolution of sample
components because the proteins are fo-cused, or “stacked,” as thin bands prior toentering the resolving gel. Second was theuse of the detergent sodium dodecyl sulfate(SDS) and reducing agents to denature pro-teins (7). SDS binds strongly to proteins atan approximate ratio of 1 dodecyl sulfatemolecule per 2 amino acid residues (8).Therefore, the negative charge/unit massratio when SDS is bound to the polypeptidechain is similar for all proteins. Third wasthe combination of the first two discoveriesemploying a simple Tris-glycine buffersystem (9). More recently, buffer combina-tions such as Tris-borate (10) and Tris-tricine (11) have improved the resolvingpower of the original methods. ModernSDS-PAGE has evolved to use microslabprecast gels (12). Precast and packaged gelsin a wide variety of gel formulations, acry-lamide percentages, thicknesses, well for-mats, and buffer systems are now commer-cially available from several manufacturers.Therefore, successful SDS-PAGE analysis ofprotein samples no longer depends on te-
dious gel casting, buffer preparation and ap-paratus set-up, but on careful sample prepa-ration and treatment prior to loading thegel. This article describes techniques andprocedures as a guide for preparation of pro-tein samples for SDS-PAGE analysis.
Sample buffer preparation
To ensure consistent and successfulPAGE analysis, the highest purity reagentsshould be used to prepare sample bufferstock solutions. After a reliable source ofelectrophoresis reagents has been identified,the vendor and buffer component chemicalsshould be maintained. High purity elec-trophoresis, Ultrol® grade, and molecular bi-ology grade reagents are available throughNovagen’s partner brand, Calbiochem.Solutions must be carefully and safely pre-pared, dated, and chemical lot numbersrecorded. Concentrated stock solutionsshould not be stored for long periods oftime. Tris base, rather than Tris-Cl, shouldbe used for buffer preparation and pH ad-justment made with HCl. Use of Tris-Cl
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ARTICLE
Preparation of protein samples for SDS-polyacrylamide gelelectrophoresis: procedures and tipsAnthony C. Grabski1 and Richard R. Burgess2—1Novagen, Inc. and 2McArdle Laboratory for Cancer Research, University ofWisconsin-Madison, Madison, WI 53706
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pensive method to maintain very low basalexpression levels of T7 RNA polymerase inthe λDE3 lysogenic expression hosts usedin the pET System. This is especially truewhen λDE3 hosts carrying pET plasmidsare grown to stationary phase. A disadvan-tage with glucose addition is that after aninitial rapid growth phase the metabolicbreakdown products of glucose will lead toacidic culture conditions and lower celldensity at stationary phase. The data pre-sented in Figure 2 demonstrate that stronginduction can be achieved from λDE3 lyso-gens in the presence of glucose for sometarget proteins. Note, however, that theoret-ically the strongest induction of T7 RNApolymerase would be expected when glu-cose is absent and cAMP levels are elevated.Accordingly, in some cases (see precedingarticle), higher target protein expression
may be observed in the absence of glucose.Overall, the optimal combination of strin-gent uninduced repression and high in-duced expression may be achieved by initialgrowth in the presence of glucose, followedby switching to medium without glucosefor induction.
Novagen’s recommendations for growthand induction of pET constructs in expres-sion hosts are based on the information pre-sented above. For innocuous proteins, anypET vector and λDE3 lysogen are suitablein a variety of media. But for proteins thatare potentially toxic to the bacterial cell, werecommend using either a pET vector witha T7lac promoter or expression hosts thatcarry the pLysS plasmid. In addition, ourgeneral advice is to avoid growing a λDE3lysogen carrying a pET plasmid to station-ary phase. If the cells must be grown to sta-
tionary phase, we recommend the additionof 0.5 to 1.0% glucose to the medium, sothat the glucose effect can be exploited toreduce basal expression.
REFERENCES1. Grossman, T. H., Kawasaki, E. S.,
Punreddy, S. R., and Osburne, M. S.(1998) Gene 209, 95–103
2. Pan, S. and Malcolm, B. A. (2000)BioTechniques 29, 1234–1238.
3. Le Grice, S. F. J., Matzura, H., Marcoli,R., Iida, S., and Bickle, T. A. (1982) J.Bacteriol. 150, 312–318.
4. Kelley, K. C., Huestis, K. J., Austen, D.A., Sanderson, C. T., Donoghue, M.A., Stickel, S. K., Kawasaki, E. S., andOsburne, M. S. (1995) Gene 15,33–36.