Vesiperhosen silkin tuotanto rekombinanttiproteiinina – olosuhteiden ja analyysimenetelmien optimointi

Caddisfly larvae silk works as an underwater adhesive. Underwater adhesion is of interest because most glues break down in water. Water-resistant glues have the potential to be applied for several applications in the fields of biomedicine and cosmetics such as biosensors and anti-aging products. In natural systems, caddisfly silk is produced in the silk glands of larvae, however, scaling this process to industrial production is inefficient and slow. Therefore, it is of interest to develop a method to produce caddisfly silk recombinantly in microbial production hosts, such as in Escherichia coli. One challenge hampering the recombinant production is that E. coli cannot perform the post-translational modification that is essential for caddisfly silk function: phosphorylation. To achieve phosphorylation, the bacterium can be engineered to produce a kinase that phosphorylates the target protein. The aim of this thesis was to optimize the expression conditions in order to improve the production yield of phosphorylated caddisfly silk in E. coli. The effect of different expression temperatures, inducer concentrations, silk types, induction timings and medias were evaluated. Furthermore, three different analysis methods for detecting phosphorylated silk were assessed. Finally, the purification method is optimized, including studying the effect of a heat precipitation step on the purity of the final product. The results show that the optimal temperature for caddisfly silk production is 20 °C, and that Diamond-Q phosphostaining and Western blotting are the most effective analysis methods. Heat shock also improved the purity of the final product, and the purification method was highly effective for isolating caddisfly silk. The results found in this research suggest that phosphorylated and non-phosphorylated caddisfly silk can be efficiently produced in E. coli, and that in addition to the optimal conditions identified in this research, further modifications of conditions can be investigated to optimize production even further.