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Applications of BBa_K5398020

In order to obtain proteins with self-healing properties, we used the pET-29a(+) vector to express TRn5 ( BBa_K5398001) ). We tried different strategies for TRn5 protein production and purification and tested its function.

Characterization

In order to obtain proteins, test suitable expression conditions, and evaluate the function of TRn4-mfp5, we chose three different expression vectors (Fig. 1)—pET-28a(+), pET SUMO, and pET-21a(+)—and tried different strategies for TRn4-mfp5 protein production and purification.

Protein Expression

We expressed the protein in E. coli BL21 (DE3) using LB medium. After incubation at 16°C for 20 hours and then at 37°C for 4 hours, we found that the protein expressed better under the 16°C for 20 hours condition, as indicated by the stronger bands in Fig. 2. This suggests that lower temperature incubation may enhance protein solubility and proper folding, resulting in improved yield.

Since there was some discrepancy in the target band size observed in the protein gel image, and the bands were not very distinct, we also tried another medium in an attempt to increase the expression level of the fusion protein. We additionally used TB medium and compared its expression efficiency with that of LB medium. We found that the bands in the TB medium were indeed thicker than those in the LB medium, indicating a slight increase in expression levels, although the difference was not significant.

We compared protein expression between the BL21(DE3) and Rosetta E. coli strains. Rosetta, derived from BL21, includes a compatible chloramphenicol-resistant plasmid that provides tRNA genes for six rare codons (AUA, AGG, AGA, CUA, CCC, GGA) that are lacking in E. coli<i/>. This modification is intended to address expression limitations associated with the high usage frequency of these rare codons in eukaryotic genes. We used the pET SUMO vector for expression. The results indicate that the protein expression level in the BL21(DE3) strain is higher compared to that in the Rosetta strain.

Fig. 4 | Comparison of fusion protein expression in E. coli strains BL21(DE3) and Rosetta.

1. Protein ladder Lanes 2-4 (BL21(DE3) LB 37℃ 4h) 2. total liquid (+IPTG); 3. supernatant (+IPTG); 4. precipitate (+IPTG); Lanes 5-7 (Rosetta LB 37℃ 4h) 5. total liquid (+IPTG); 6. supernatant (+IPTG); 7. precipitate (+IPTG).

Fig. 4 | SDS-PAGE of purified fusion protein TRn4-mfp5 (35.4 kDa) using vector pET SUMO.

Lane 1: Protein - Binding buffer;
Lane 2: 20 mM imidazole and 8 M urea elution; Lane 3: 50 mM imidazole and 8 M urea elution; Lane 4: 150 mM imidazole and 8 M urea elution; Lane 5: 300 mM imidazole and 8 M urea elution; Lane 6: 500 mM imidazole and 8 M urea elution; Lane 7: Supernatant; Lane 8: Impurities; Lane 9: Protein ladder.

Protein Purification

We obtained protein samples of TRn4-mfp5 by freezedrying 24 h (Fig. 6). The final yield was about 25 mg/L bacterial culture.

Fig. 6 | The protein sample freeze-dried by a lyophilizer.

Next, we dissolved protein samples in Buffer A (10 ml 20 mM Tris pH8) to reach 0.3 mg/mL, and conduct adhesive ability tests on the fusion protein(Fig. 7). 200 μL of the protein solution was applied, and the pipette tip was placed on a plastic Petri dish lid. After incubation at 37°C for 8 hours, the pipette tip successfully adhered.

Fig. 7 | Adhesive Ability Test of Fusion Protein on Plastic Surface

Reference

<p>[1] Jung, H., Pena-Francesch, A., Saadat, A., et al. Molecular tandem repeat strategy for elucidating mechanical properties of high-strength proteins [J]. <i>PNAS, 2016, 113(23): 6478–6483.

[2] Zhang, C., Wu, B., Zhou, Y., et al. Mussel-inspired hydrogels: from design principles to promising applications [J]. Chem Soc Rev, 2020, 49: 3605-3637.

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