ACES Promoter Selection Vector Technical Brief
Sheldon E. Broedel, Jr., Ph.D. and Sharon M. Papciak, Ph.D.
Athena Environmental Sciences, Inc., Baltimore, MD.
In the early days of producing proteins in E. coli, limitations to transcription initiation were believed to
lead to lower expression levels.1 This was often true and the historical result was the almost exclusive
construction and deployment of expression vectors which carried strong promoters. These include the phage
promoters T7 and T5, the synthetic promoters tac and trc, and the arabinose inducible ara.2 The T7
IPTG-inducible system has become the dominant expression system currently employed. However, the use of a
strong promoter, which leads to hyper-expression levels, can adversely effect recombinant protein expression.3,4
The most frequent and challenging problem encountered with hyper-expressed proteins is that the proteins can
accumulate as inclusion bodies. These insoluble particles require that the target protein be denatured and then
refolded in order to recover soluble protein.5 The process of refolding proteins can be a daunting task. The
optimum conditions for recovering active protein requires the evaluation of several variables, is not always
possible and, when successful, often results in poor yields. The reasons for proteins accumulating as inclusion
bodies are varied, involving both the intrinsic properties of the protein as well as interactions with host
proteins. One approach used to limit the accumulation of inclusion bodies involves lowering the level of expression.
This is done by decreasing the culture temperature during expression, decreasing the amount of inducer (if a
titratable promoter is used), employing alternative strains (with higher levels of chaperone proteins or other
accessory proteins), or using weaker promoters. None of the approaches by themselves will give the best solution
and maximum production of soluble protein still requires the use of matrix analyses to identify the optimum
expression conditions. The first three alternatives are relatively easy to control experimentally. The latter,
however, currently requires the subcloning of the target gene sequences into a small number of alternative
expression vectors which carry different inducible promoters. Thus, the ability to easily examine a range of
promoter strengths to identify the best for the expression of a particular protein has been currently limited.
Figure 1. Plasmid map of pAES25.
To facilitate the construction of promoter variants that yield the maximum level of soluble active target
protein we designed a simplified method for creating a library of promoter mutants. The coding sequence for
the desired protein is first inserted into the expression vector pAES25 (Fig. 1). Promoters with different
strengths that direct the expression of the target protein are made from a set of specifically designed PCR
primers. These are made using an inverse PCR technique that introduces random sequences within the promoter
region. The promoter variant that yields maximum levels of soluble, active protein is identified by screening
the library. Since the promoter selection is performed in the expression vector pAES25, no further subcloning
is needed to express and purify the target protein.
Figure 2. Analysis of the PCR product. A 5 μl sample of each amplification reaction was loaded onto a 0.7% agarose-TBE gel and electrophoresed.
To demonstrate the potential utility of this technique, the coding sequence for the Ptilosarcus green fluorescent
protein (Prolume, Ltd.) was subcloned into pAES25 at the SalI site. This resulted in a plasmid, pAES25-PtGFP,
where the MCS was left intact and the GFP was expressed beginning at the AUG start codon that is located just
upstream of the BamHI site. Expression of the Pt-GFP protein from this plasmid results in the accumulation of
the protein into inclusion bodies; however, in vivo fluorescence can still be detected. Purified pAES25-PtGFP
was then used as a template for amplification with Primer A (N17GATTCAATTGTGAGCGG) and Primer B (N17ATTTTTTATGATTTCTCGAG).
Two 100 μl reactions containing 10 ng of plasmid DNA, 20 pmoles of each primer, 200 μM dNTPs and 0.5 Unit Hot
Star DNA Polymerase (Qiagen) was heated to 95°C for 15 min and then subjected to 20 cycles of 95°C, 30s; 60°C,
30s with -0.5°C per cycle; 72°C, 5 min followed by 15 similar cycles except that the annealing temperature was
50°C and the final extention temperature at 72°C was extended by 10 minutes. before chilling to 4°C. The
resulting amplification product was analyzed by agarose gel electrophoresis (Fig. 2) and then gel purified using
the Qiaex II Gel Purification Kit (Qiagen) according the manufacturer?s protocol. The purified DNA was recovered
in 40 μl of 10 mM Tris-Cl, pH 8.5. To ligate the vector, a 2 μl aliquot was phosphorylated in a 10 μl reaction
(20 Units kinase in 50 mM Tris-Cl pH 7.5, 10 mM MgCl2, 5 mM DTT, 50 pmoles ATP), heated at 75°C for 2 min.,
chilled on ice for 5 min. and ligated by adding 1 Weiss unit of T4 DNA ligase. After 1 hour at 22°C, the DNA
was introduced into competent cell strain HMS174 and KmR transformants were selected.
Figure 3. Accumulation of GFP in 93 promoter mutants. The increase in fluorescence was measured
in cultures after the addition of IPTG to 1 mM. Fluorescence was measured at 30 min. intervals for 8 hours
using a λex of 485 nm, λem of 535 nm, gain 82, 3 flashes, and 40 μs integration. The parent strain harboring
pAES25-PtGFP showed high GFP levels before induction which resulted in fluorescence above the detection limit
of the plate reader.
|Relative Fluorescence Units per OD600 at 4 hours post-induction
Table 1. Wells A1 through E12 are the individual isolates 1 to 93. Well F12 is pAES25-PtGFP, well G12 is
pAES25 and well H12 is a medium blank. RFU values of the isogenic parent strain were subtracted.
Isolates expressing the GFP protein were identified by inducing expression of 93 independent transformants. Each of
93 colonies were used to inoculate 0.2 ml of Turbo Broth (AthenaES) in a 96-well microplate. Strains harboring
pAES25 and pAES25-PtGFP were used as parental controls. The cultures were incubated at 37°C overnight with shaking
using a GENIOS Fluorescence Microplate Reader. A 10 μl aliquot of each culture was subcultured into 0.2 ml of fresh
medium and the microplate incubated at 37°C for 3 hours. GFP expression was induced by the addition of 1 mM IPTG
and the level of fluorescence measured at 30 min intervals for 8 hours. Figure 3 shows the kinetics of fluorescence
accumulation after induction. Of the 93 isolates selected 10 showed significant levels of GFP accumulation with several
others showing relatively slight increases in fluorescence. The induction experiment was repeated except analysis of the
total fluorescence produced after 4 hours induction was determined for each culture and normalized to culture density.
Table 1 and Figure 4 show that relative fluorescence units (RFU) per OD ranged from a low of 147 (F2, #14) to a high of
23,773 (A1, #1). Most isolates had RFU/OD values below 850 whereas 10 isolates had RFU levels that were more than 5-fold
higher. The 83 isolates exhibiting low fluorescence are likely the result of leaky transcription inherent in plasmids rather
than transcription initiating at the modified promoter region. The 10 isolates which exhibited inducible expression from the
promoter variants showed a 5-fold range in GFP levels. Their fluorescent levels were about 2-fold less than the parent plasmid.
Microscopic examination revealed that none of the promoter variants yielded visible inclusion bodies whereas the strain
harboring the parent plasmid did. These results suggest that by reducing the transcription rates the amount of soluble
protein can be increased. Further quantitative analysis of the amount of Pt-GFP produced should confirm these initial
Figure 4. Fluorescence levels at 4 hours post-induction. ?P? is the parent strain HMS174/pAES25-PtGFP.
Decreasing the expression levels of recombinant proteins can alleviate the accumulation of a protein in an insoluble
state. Similarly, the adverse effects of over expressing proteins that are toxic to the host, can be mitigated by
reducing the expression level of the recombinant protein. Lowering the transcription rates is one approach for decreasing
expression levels. The vector described here, provides a simple and rapid method for identifying a promoter that has the
transcription initiation rates which are best suited for the production of a soluble or toxic protein when the protein
is otherwise difficult to obtain using the strong promoters carried on most expression vectors. This tunable expression
system can be used in conjunction with other techniques to maximize the accumulation of the desired protein.
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