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 thermal shift assay 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.

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 band was visible at the height above 30 kDa after 3h. 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.