Thermal and acidic extraction methods are based on innate silk properties: thermal stability and resistance to acid.High temperature and concentrated acid cause precipitation of expression host proteins, whereas silk proteins remain soluble.In our previous study, we investigated the relationship between purification method and cytotoxicity of the Ma Sp1-based bioengineered silk proteins (15X and 6X).We showed that thermal and acidic extraction methods yield pure, non-toxic and non-immunogenic soluble silk variants when tested over a wide range of concentrations.These methods yield pure silk protein with high efficiency.However, as the greatest advantage, both methods circumvent the need for a tag sequence, which is ultimately favorable for biomedical applications.Although native spider silk is difficult to obtain in a pure form and in sufficient quantities, the development of recombinant spider silk production techniques and purification methods successfully solved the accessibility problem and paved the way for further research.Bioengineered silks produced in heterologous hosts (most commonly Escherichia coli) can form aggregates within the cell (inclusion bodies) and can be secreted in a soluble form to the cytoplasm or outside the cell to the culture media.
Despite the simplicity and robustness of this technique, the proteins obtained by this method possess an affinity domain that may alter the protein properties, disrupt their function or cause protein cytotoxicity.
A novel, tag-free, bioengineered spider silk named MS2(9x) was constructed.
It is a 9-mer of the consensus motif derived from Ma Sp2–the spidroin of Nephila clavipes dragline silk.
Using this method, it has been found that the protein concentration, phosphate concentration and p H of potassium phosphate play a role in the particle formation process.
Yet, a great number of factors that can affect the sphere formation process and the spheres’ properties are not well understood.