Team:TU Darmstadt/Results/Scaffold

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Scaffold

We started by cloning several constructs to provide the registry with different scaffold variants and its single parts. For this purpose we constructed a library of various combinations of the single domains in pSB1C3. Subsequently we were able to modify the scaffold at its C-terminus with a Strep- and a His-tag.

Furthermore, a system for the production of the primarily developed scaffold was established that contains one of each domain. The codon-optimized scaffold sequence was cloned into a pET21a+ expression vector with a C-terminal His-Tag. 

The test expression in a successfully transformed BL21(DE3) strain in LB medium showed a successful production of the scaffold (Figure 1(a)). The cells quickly showed a high level of expression after induction, only a slight increase of the band intensity was detected afterwards.

Figure 1: SDS PAGE for the analysis of the production of the scaffold in LB medium. a) Expression of 2 colonies. The samples before induction (Vi) and the progression over 4 h is depicted. A band of increasing intensity at expected height exists for both colonies. b) Analysis of the IMAC purification: the pellet (Pel), the supernatant after centrifugation (Üs), the flow-through (Df) and 5 eluted fractions were analysed.

The SDS PAGE (Figure 1 (b)) for the analysis of the purification of the scaffold via a His-tag showed, that the supernatant of the centrifugation contained a majority of the produced protein, although a distinct band was also visible for the pellet. The protein is efficiently bound to the column, so that the expected band is no longer visible in the flow-through. The protein was detected in fractions 11 and 12 of the Ni-NTA-purification and was accordingly eluted at an imidazole concentration of 100 - 200 mM . 

A photometric measurement at 260 nm determined a yield of 3 mg of protein in both fractions. This sums up to a total yield of 6 mg/L for the described method, whereas a quantitative estimation of the remaining amount in the pellet was not possible. Multiple bands are visible in fraction 11, fraction 12 is less contaminated.

Subsequently this enabled us to proceed with some stability tests of the three-domain scaffold to screen for the optimal working and storing conditions of the molecule. The influences of urea, KCl and pH changes on the stability of the scaffold were observed (Figure 2). 

Urea does not cause denaturation at concentrations below 500 mM at low temperatures. The protein remains stable in 50 mM Tris at pH 8 at < 26°C (Figure 2 (a)). The additionally tested concentrations of 5 and 8 M are not presented since the protein was already denatured at 15°C. During the experiment with KCl, it became clear that the protein gained stability with increasing concentration (Figure 2 (b)). While denaturation started at 25°C with low KCl concentration, the stability rapidly increased, approaching a denaturation temperature of 40°C asymptotically when the KCl concentration was > 500 mM. A pH optimum was found at about 7 - 7.5 (Figure 2 (c)). Below pH 7, the thermal stability decreased rapidly. The protein was not correctly folded at pH 5.5 – 6 even below 15°C, while pH values above 7.5 showed similar, yet less drastic results.

Figure 2: Results of the stability tests of the scaffold. a) Graph of denaturation temperature at different urea concentrations in solutions of 50 mM Tris at pH 8. b) Graph of denaturation temperature at different KCl concentrations at pH 7. c) Graph of denaturation temperature at different pH values. Stability shows a maximum around pH 7.

The results match an expected behavior. The denaturing urea lowers the scaffold’s stability at higher concentrations while high salt concentrations support its structure. The stability curve at different pH values is similar to the expectation. The pI of the molecule lies between 5.8 and 6.2, at these pH values the protein is expected to show the minimal solubility. Accordingly there is less solved protein to interact with the dye and a lower or non-existent intensity through denaturation. At pH > 7.5 the stability of the protein drops, since the amount of negatively charged amino acid rests increases and the amount of positively charges decreases respectively. This leads to stronger repulsion inside the molecule and lowers the energy that is necessary for denaturation.

The experiments showed that the scaffold protein is heat labile, which explains the suggested expression temperature of 30° C from Dueber (Dueber et al. 2009) and could be a hint, why lower temperatures might prevent the formation of inclusion bodies and thereby increase the yield.

For an additional usage of the scaffold protein we attached a cysteine to the C-terminal His-tag. The mutant was fitted in pSB1C3.

After the addition of the Cystein the mutant was also expressed in the pET21a+ system. An experiment was performed in order to provide the basis for a successful production of the Scaff-His-Cys variant. For that purpose, the product was examined by PAA gel electrophoresis (Figure 3).

The gel was load with a sample of the pre-culture and the cells prior as well as several hours after induction. A clear band was visible at the height above 30 kDa. The scaffold with His-tag has a size of 31.17 kDa. As a consequence, the band appearing after induction can be considered as the correct protein.




Fig. 3: SDS-PAGE of the expression control of the scaffold protein with cysteine in E. coli BL21(DE3) cells. Next to the marker (BLUEplus perstained Protein Ladder, Biomol), cell lysates were applied of the pre-culture and of cells before and after several hours of induction. A clear overproduction of the protein is present at 4h after induction. The scaffold protein has a size of about 31.17 kDa.

Due to the presence of the scaffold protein in the lane of the pre-culture, it can be concluded that the inducible promoter is leaky. A clear overproduction of the scaffold protein is only reached after an incubation time of 4h.

References

John E. Dueber, Gabriel C. Wu, G. Reza Malmirchegini, Tae Seok Moon, Christopher J. Petzold, Adeeti V. Ullal, Kristala L.J. Prather, und Jay D. Keasling, "Synthetic protein scaffolds provide modular control over metabolic flux". Nature biotechnology, 27: 753 - 759, 2009.