Difference between revisions of "Part:BBa K5301005"

Basic Silinker (BS) is a novel recombinant protein that efficiently attaches to the surface of silicon dioxide. The presence of mSA often leads to the formation of inclusion bodies, increasing the difficulty in purification. To achieve efficient expression of Basic Silinker and minimize the formation of inclusion bodies, we screened the induction conditions using IPTG. We set up a gradient of five IPTG concentrations: 0mM, 0.1mM, 0.25mM, 0.5mM, and 1mM. The results showed that the optimal protein expression was achieved with a concentration of 1mM. Furthermore, we tested two temperature gradients for induction: 37°C and 16°C. At 37°C, the protein mainly formed inclusion bodies rather than soluble proteins. Therefore, we determined that 16°C was the effective induction temperature. To facilitate proper folding of mSA and reduce inclusion body formation, we modified the protein buffer by incorporating biotin. The binding of biotin to mSA helps with the correct folding of the Basic Silinker protein, minimizing the formation of misfolded inclusion bodies. As a result, we obtained soluble proteins that could be extracted from the supernatant. The formulation of the buffer and experimental procedures can be found in our protocol.