Part:BBa_K5298001
The 2xKLV-Mfp5 sequence can encode ordered amyloid proteins and disordered Mfp5 proteins, combining
This part selects the zipper-forming sequence from human Aβ amyloid protein due to its strong tendency to self-assemble into stable β-crystals under aqueous conditions. Then, a flexible sequence from the Nephila clavipes dragline spider silk protein MaSp1 is chosen to link multiple zipper-forming sequences together, allowing multiple zipper-forming sequences within a single protein to fold into β-sheets. The amyloid and spider silk sequences are repeated twice to achieve sufficient chain length, which is crucial for favorable material strength. To impart surface adhesion to the protein material, Mytilus galloprovincialis 5 (Mfp5) is then added to the C-terminus of the repeated sequences. In addition to surface adhesion, the disordered Mfp5 chains can also utilize dihydroxyphenylalanine (DOPA) and other amino acid residues to induce intermolecular interactions between hybrid proteins, thereby enhancing the cohesion of the material. Overall, the combination of ordered (amyloid) and disordered (Mfp5) sequences is expected to form β-crystals and amorphous domains, respectively, and the extensive intermolecular interaction network between the silk-amyloid-Mfp5 molecules (termed 2xKLV-Mfp5) can produce strong and tough semi-crystalline protein hydrogels that also exhibit underwater adhesion through surface-exposed DOPA residues in Mfp5.
Sequence and Features
- 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]
Tongji-China 2024: Characterization of sticky hybrid proteins
To express a fusion protein with adhesive properties, we first designed a plasmid using the pET28a vector as the backbone. This gene construct was then sent to Genewiz for synthesis.
Subsequently, we extracted the target plasmid from the stab cultures returned by the company and transformed it into the DE3 Escherichia coli strain to obtain monoclones, preparing for the induction of the expression of the target protein.
To verify whether the target plasmid would express the target protein in the DE3 strain, we first conducted a preliminary experiment to explore the induction conditions.
Preliminary Experiment:
1. We first picked a single colony of the transformed DE3 and inoculated it into 5 mL of LB medium for overnight culture at 37°C to generate a seed culture.
2. Three 200 mL conical flasks were filled with 50 mL of LB medium each, and 1.5 mL of the seed culture was inoculated into each flask. Two flasks were incubated at 37°C, while one flask was incubated at 30°C for 7-8 hours. At this point, the OD600 value was measured to be approximately 1.5. From each flask, 500 μL of bacterial suspension before induction was withdrawn, and then 1 mM IPTG was added for protein induction. After 6-7 hours of incubation at 37°C, 500 μL of bacterial suspension from each flask was withdrawn, and along with the pre-induction samples, they were subjected to SDS-PAGE analysis. The results indicated the expression of the target protein.
3. To determine whether the protein was present in the supernatant or the precipitate after cell lysis, we collected the bacterial cells, resuspended them, and performed ultrasonic disruption followed by centrifugation. Subsequently, we performed SDS-PAGE on total bacterial cells, the supernatant, and the precipitate. The final results indicated that the protein was present in the supernatant.
Given the promising preliminary experimental results, we proceeded with the formal experiment.
1. The seed culture was inoculated into 50 mL of LB medium and shaken overnight at 37°C.
2. Two 2 L large conical flasks were prepared, each containing 500 mL of LB medium. The well-shaken 50 mL bacterial suspension was divided equally into the two flasks, and the flasks were shaken at 37°C for 2-3 hours until the OD600 reached 1.5-1.8. Subsequently, 1 mM IPTG was added to induce protein expression for 10-12 hours.
3. The bacterial suspension was first centrifuged to discard the supernatant and collect the bacterial cells. The cells were resuspended and subjected to ultrasonic disruption for 1 hour, followed by centrifugation to collect the supernatant.
4. Since the target protein has a histidine tag, nickel affinity chromatography was employed for purification. The process involved equilibration, sample loading, re-equilibration, washing to remove impurities, and finally elution to purify the target protein.
5. Samples were collected from each step of the purification process and subjected to SDS-PAGE analysis to observe the results.
We have expressed the fusion protein Mfp5-Aβ amyloid-MaSp1 in
Escherichia coli and purified the target protein from the bacterial precipitate. We have demonstrated the correct purification of the protein using SDS-PAGE. After catalyzing tyrosine to dopamine using tyrosinase, our protein will exhibit the expected adhesive properties, achieving the mixing and final completion of the AMS nail adhesive layer.
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