Part:BBa_K5298001:Design
The 2xKLV-Mfp5 sequence can encode ordered amyloid proteins and disordered Mfp5 proteins, combining
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 5
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 67
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
Due to the repetitiveness of the gene, codon optimization of the repeated KLV sequences is necessary during gene synthesis. Furthermore, when expressing this protein in Escherichia coli BL21(DE3), we initially assumed that the expression level of the protein in the bacterial precipitate would be significantly higher than that in the supernatant, owing to the protein's strong adhesiveness and tendency to self-assemble into stable crystals, resulting in its presence in inclusion bodies and subsequent precipitation during centrifugation. However, in actual experiments, we were unable to purify sufficient amounts of protein from the precipitate. Consequently, we abandoned the previous direct purification approach and instead first confirmed the protein's localization using SDS-PAGE gels before culturing the bacterial solution in a larger system to extract and purify large quantities of protein. Shifting to extraction from the supernatant required a longer time compared to precipitation extraction. Therefore, we patiently cultured the bacteria in a larger system for an extended period and ultimately succeeded in extracting the target fusion protein from the supernatant.
Source
This component comes from the literature:Kim E, Jeon J, Zhu Y, Hoppe ED, Jun YS, Genin GM, Zhang F. A Biosynthetic Hybrid Spidroin-Amyloid-Mussel Foot Protein for Underwater Adhesion on Diverse Surfaces. ACS Appl Mater Interfaces. 2021 Oct 20;13(41):48457-48468. We learned the design idea of adhesion protein from this literature, and changed the sequence to design the 2xKLV-mfp5 element.