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Latest revision as of 02:52, 29 October 2011

SgsE from Geobacillus stearothermophilus NRS 2004/3a

Oblique structure of S-layer lattices.

SgsE monomers are naturally assembled in a lattice with oblique symmetry (p2) (compare figure on the right) exhibiting a well-defined periodicity and distances of 9.4 – 11.6 nm between the proteinaceous subunits. The S-layer protein SgsE of Geobacillus stearothermophilus NRS 2004/3a consists of 903 amino acids, including a 30 amino acid signal peptide (SLH-domain) at the amino-terminus. The carboxy-terminal part of SgsE is the larger part of the protein, encoding the self-assembly information. The protein is formed by the sgsE gene, has a calculated mass of 93.7 kDa and an isoelectric point of 6.1. When isolated, SgsE maintains its ability to self-assemble, and in dependence of salt concentration, duration of dialysis to remove the detergent and its amino acid sequence, it builds up five types of self-assembly products. These products are formed like flat sheets and cylinders ([http://onlinelibrary.wiley.com/doi/10.1002/smll.200700200/abstract Schäffer et al., 2007]).

Expression in E. coli

The sgsE gene under the control of a T7 / lac promoter (<partinfo>K525303</partinfo>) was fused to mCitrine ([http://partsregistry.org/Part:BBa_J18931 BBa_J18931]) using Freiburg BioBrick assembly for characterization experiments.

The SgsE|mCitrine fusion protein was overexpressed in E. coli KRX after induction of T7 polymerase by supplementation of 0.1 % L-rhamnose and 1 mM IPTG using the autoinduction protocol by Promega.

Figure 1: Growth curve of E. coli KRX expressing the fusion protein of SgsE and mCitrine with and without induction, cultivated at 37 °C in autoinduction medium with and without inductor, respectively. A curve depicting KRX wildtype is shown for comparison. After induction at approximately 4 h the OD₆₀₀ of the induced <partinfo>K525305</partinfo> clearly falls behind when compared to the uninduced culture. Both cultures grow significantly slower than KRX wildtype probably due to a leaky promoter and metabolic stress by the high copy plasmid.

Figure 2: RFU to OD₆₀₀ ratio of E. coli KRX expressing the fusion protein of SgsE and mCitrine with and without induction. A curve depicting KRX wildtype is shown for comparison. After induction at approximately 4 h the RFU to OD₆₀₀ ratio starts to rise in the induced culture. Compared to the uninduced culture the ratio is roughly four times higher. The KRX wildtype shows no variation in the RFU to OD₆₀₀ ratio.

The SgsE gene under the control of a T7 / lac promoter (<partinfo>K525303</partinfo>) was fused to firefly luciferase (<partinfo>K525999</partinfo>) using Freiburg BioBrick assembly for characterization experiments.

The SgsE|luciferase fusion protein was overexpressed in E. coli KRX after induction of T7 polymerase with 0.2 % L-rhamnose and induction of lac operator with 1 mM IPTG.

The following cultivation was carried out in a [http://www.gmi-inc.com/BioEngineering-KLF-Small-Laboratory-Fermenter.html#product_desc Bioengineering KLF] bioreactor with Bioengineering DCU and software. A sequencer which automatically pumped an inducer solution after 4 h cultivation time to start protein expression was implemented. Other parameters were:

• Medium: HSG medium with 20 mg L^-1 chloramphenicol
• Culture volume: 2.5 L
• Starting OD₆₀₀: 0.4
• DO: 60 % airsaturation (controlled with stirrer cascade starting with 200 rpm)
• pH: 7.0 (controlled with 20 % phosphoric acid and 2 M NaOH)
• Antifoam: BASF Pluronic PE-8100
• Induction solution: 0.2 % L-rhamnose and 1 mM IPTG

The following figure shows the expression of the SgsE | luciferase S-layer fusion protein <partinfo>K525311</partinfo> in E. coli KRX in HSG medium with autoinduction sequencer as described above. Optical density, activity of the fused luciferase, dissolved oxygen and agitator speed are plotted against the cultivation time.

Fig. 3: Bioreactor cultivation of E. coli KRX expressing the fusion protein SgsE :: luciferase <partinfo>K525311</partinfo> under the control of a T7 / lac promoter. Induction with 1 mM IPTG and 0.2 % L-rhamnose after 4 h controlled by sequencer. Cultivation in [http://www.gmi-inc.com/BioEngineering-KLF-Small-Laboratory-Fermenter.html#product_desc Bioengineering KLF] bioreactor with 2.5 L HSG medium with 20 mg L^-1 chloramphenicol starting with OD₆₀₀ = 0.4. Dissolved oxygen was regulated to 60 % airsaturation (controlled with stirrer cascade starting with 200 rpm) and the pH to 7.0 (controlled with 20 % phosphoric acid and 2 M NaOH). The dissolved oxygen sensor had to be recalibrated after about 140 min.

Purification of SgsE fusion protein

Purification of SgsE | mCitrine without His-tag

As observed in the analysis of the cultivations with expression of SgsE | mCitrine fusion proteins, these proteins form inclusion bodies in E. coli. Inclusion bodies have the advantage that they are relatively easy to clean-up and are resistant to proteases. The first purification step is to isolate and solubilize the inclusion bodies. This step is followed by two filtrations (300 kDa UF and 100 kDa DF/UF) to further concentrate and purify the S-layer proteins. After the filtrations, the remaining protein solution is dialyzed against ddH₂O for 18 h at 4 °C in the dark. The dialysis leads to a precipitation of the water-insoluble proteins. After centrifugation of the dialysate, the water-soluble S-layer monomers remain in the supernatant and can be used for recrystallization experiments.

The fluorescence of the collected fractions of this purification strategy is shown in the following figure 4:

Figure 4: Fluorescence of collected fractions during purification of <partinfo>K525305</partinfo> fusion protein.

A huge amount of protein is lost during the purification, especially after the centrifugation steps. The fluorescence in the urea containing fractions is lowered due to denaturation of the fluorescent protein. Some fluorescence could be regenerated by the recrystallization in HBSS. This purification strategy is very simple and can be carried out by nearly everyone in any lab, being the first step to enable do it yourself nanobiotechnology.

Purification of SgsE | mCitrine with His-tag

By fusing the SgsE | mCitrine with a C-terminal [http://partsregistry.org/Part:BBa_K157011 His-6-tag] the S-layer protein could be simply purified by using a denaturating His-tag affinity chromatography. This purification strategy has the advantage that no time-consuming and complex inclusion body purification and filtration is necessary to decrease the amount of native E. coli proteins. Additional to the simplification a higher purity of the S-layer protein could be reached.

This purification was performed using the SgsE fusion protein containing a N-terminal mCitrine,for identification and a C-terminal His-6-tag. The SDS-PAGE gel (Fig. 5) and the fluorescence (Fig. 6) in the collected fractions showed that the majority of fusion protein was eluated with a imidazole concentration of approx. 50 mM. There was also fluorescence measurable in the flowthrough and wash fractions. This indicates that the used 1 mL HisTrap FF crude (GE Healthcare) was overloaded or the protein was bound weakly at to the affinity matrix. The resulting purity in the elution fraction, the saving of time and the simplicity makes this procedure the prefered purification method.

Fig. 5: SDS-PAGE gel of a denaturating immobilized metal affinity chromatography (IMAC) of a SgsE fusion protein containing a N terminal mCitrine and a C-terminal His-6-tag. A 1 mL HisTrap FF crude by GE Healthcare (Ni-NTA) was used for purification. The binding buffer contained 10 mM imidazole and the elution buffer (buffer B) 500 mM imidazole. The bound proteins were eluated by a stepwise rising imidazole gradient (10 % buffer B per step). The SgsE fusion protein (111,2 kDa) was eluated in the 10 % buffer B fraction in a high purity.

Fig. 6: Fluorescence in the fractions of a denaturating immobilized metal affinity chromatography (IMAC) of a SgsE fusion protein containing a N terminal mCitrine and a C-terminal His-6-tag. A 1 mL HisTrap FF crude by GE Healthcare (Ni-NTA) was used for purification. The binding buffer contained 10 mM imidazole and the elution buffer (buffer B) 500 mM imidazole. The bound proteins were eluated by a stepwise rising imidazole gradient (10 % buffer B per step). The SgsE fusion protein (111,2 kDa) was eluated in the 10 % buffer B fraction in a high purity.

Final purification strategies for SgsE | mCitrine

Strategy with His-tag

Scheme of purification strategy for SgsE (fusion) proteins with His-tag:

First, SgsE is expressed in E. coli under the control of a T7 / lac promoter for separation of growth and production phase due to metabolic stress of the S-layer expression. Because the SgsE protein is forming inclusion bodies in E. coli, the cells were mechanically disrupted (Sonification on ice) in binding buffer containing 6 M urea. After centrifugation the supernatant is loaded as sample onto a nickel-nitrilotriacetic acid (Ni-NTA) metal-affinity column. The S-layer containing elution fraction of the denaturing His-tag affinity chromatography is afterwards dialysed against water. This leads to the precipitation of water-insoluble proteins. The supernatant contains the monomeric SgsE solution.

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Strategy without His-tag

Scheme of purification strategy for SgsE (fusion) proteins without His-tag:

First, SgsE is expressed in E. coli under the control of a T7 / lac promoter for separation of growth and production phase due to metabolic stress of the S-layer expression. Because the SgsE protein is forming inclusion bodies in E. coli, an inclusion body purification with urea follows the cell lysis. The S-layers are further concentrated and purified by two ultrafiltration / diafiltration steps (300 kDa and 100 kDa) and afterwards dialysed against water leading to the precipitation of water-insoluble proteins. The supernatant contains the monomeric SgsE solution.

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Purification of SgsE | luciferase fusion protein

After the analysis of cultivations with expression of SgsE | luciferase fusion proteins, different cell fractions were analyzed. It could be seen that the proteins form inclusion bodies in E. coli but that there are some soluble proteins left. This has the advantage that the proteins carrying an enzyme as fusion proteins do not have to be treated with denaturating agents like urea which destroys the enzyme (data not shown).

To capture the protein from the cell lysate an ion exchange chromatography (IEX) was carried out (binding with pH 7.0, 25 mM NaCl, quaternary amine beads, elution with 100 mM NaCl). A lot of protein was found in the flow-through. When concentrating and rebuffering the proteins with PES (polyethylene sulfone) membranes a lot of protein was lost. The S-layer proteins stuck to the membrane. Some could be removed again from the membrane after cutting out the filter and incubate it in ddH₂O over-night. This problem has to be kept in mind when using this S-layer.

The results of the purification approach are shown in Fig. 7:

Fig. 7: Luminescence corrected with the volume of the collected fraction plotted against the fraction during purification of <partinfo>K525311</partinfo>. Abbreviations: s.n. = supernatant, F = filtrate, R = retentate, FT = flow through, E10 = elution with 100 mM NaCl.

The purification strategy has to be improved. The inclusion bodies cannot be purified because urea damages the luciferase irreversible (data not shown). The loss due to adsorption of the SgsE | luciferase fusion protein to PES membranes could be avoided by using different membranes. The binding conditions of the IEX have to be improved as well. Anyway, the idea behind this purification strategy could be a starting point for a better strategy. Possibilities for improvement are:

• Different membranes for ultra- / diafiltration
• Other binding conditions for the IEX capturing step (higher pH)
• Hydrophobic interaction chromatography as purification step after IEX (works in general, data not shown)
• Size exclusion chromatography (SEC) for polishing

Immobilization behaviour

After purification, solutions of monomeric SgsE S-layer proteins can be recrystallized and immobilized on silicon dioxide beads in HBSS (Hank's buffered saline solution). After the recrystallization procedure the beads are washed with and stored in ddH₂O at 4 °C in the dark. The fluorescence of the collected fractions of a recrystallization experiment with <partinfo>K525305</partinfo> are shown in Fig. 8. 100 mg beads were coated with 100 µg of protein. The figure shows, that not all of the protein is immobilized on the beads (supernatant fraction) but the immobilization is quite stable (very low fluorescence in the wash). After the immobilization, the beads display a high fluorescence indicating the binding of the SgsE | mCitrine fusion protein.

Figure 8: Measured fluorescence of collected fractions of immobilization of purified <partinfo>K525305</partinfo> on silica dioxide beads (n = 3, 100 mg mL^-1 SiO₂, time of recrystallization: 4 h).

Optimal bead to protein ratio for immobilization

To determine the optimal ratio of silica beads to protein for immobilization, the degree of clearance ϕ_C in the supernatant is calculated and plotted against the concentration of silica beads used in the accordant immobilization experiment (compare Fig. 9):

The data was collected in three independent experiments. The fluorescence of the samples was measured in the supernatant of the immobilization experiment after centrifugation of the silica beads. The fluorescence of the control was measured in a sample which was treated exactly like the others but no silica beads were added. 100 µg protein was used for one immobilization experiment. The data was fitted with a sigmoidal dose-response function of the form.

with the Hill coefficient p, the bottom asymptote A1, the top asymptote A2 and the switch point log(x₀) (R² = 0.874).

The fit indicates that a good silica concentration for 100 µg of protein is 150 - 200 mg mL^-1. This set-up leads to saturated beads with low waste of protein. So a good protein / bead ratio to work with is 5 - 7 * 10^-4.

Figure 9: Degree of clearance of the fluorescence in the supernatant plotted against the concentration of silicium dioxide beads used to immobilize <partinfo>K525305</partinfo> (n = 3). Data is fitted with dose-reponse function (R² = 0.874).

Effect on enzyme stability

To test whether the S-layer protein enhances the half-life of the firefly luciferase, immobilization experiments were carried out. After the IEX described above, the elution fraction was immobilized on silicon dioxide beads or just diluted with HBSS buffer which is used for the immobilization / recrystallization of SgsE S-layer proteins. The immobilization is carried out at room temperature for 4 h. It could be seen that the luciferase activity nearly expired during this time (in the positive and the negative control). So the S-layer SgsE could not stabilize the luciferase at room temperature. The results of this experiment is shown in the figure below:

Fig. 10: Results of immobilization experiments with <partinfo>K525311</partinfo>. The stability of the luciferase activity at room temperature is decreasing quickly.