Part:BBa_K5398001

TRn5

Usage and Biology

This part codes for the biosynthetic proteins with five tandem repeats of the squid-inspired building block (TRn5). These high-strength synthetic proteins have advantages over other self-healing materials, in terms of healing properties (2-23 MPa strength after 1 s of healing), creating great opportunities for bioinspired materials design, especially in self-healing materials for soft robotics and personal protective equipment.

The tandem repeat polypeptides of TRn, driven by their segmented amino acid sequences, selfassemble into supramolecular β-sheet-stabilized networks (Fig. 1). It's proved that there exists a positive correlation between the number of repeat units and self-healing properties of squid-inspired proteins, which means the more repeat units the proteins have, the better self-healing properties it will be.

Fig. 1 The sequence and structure of squid-inspired biosynthetic proteins.

In our project, we used TRn5 as special materials to realize self-healing.

Characterization

Protein expression

In order to obtain proteins with self-healing properties, we used the pET29a(+) vector to express TRn5. We tried different strategies for TRn5 protein production and purification and tested its function.

We expressed the protein in E.coli BL21 (DE3) using LB medium. After incubation at 37℃ for 5h and 30℃ for 9 h, respectively, we found that most TRn5 (17.58 kDa) existed in precipitation and the TRn5 expression level at two temperatures had little difference (Fig. 2).

Fig. 2 SDS-PAGE of expression products of TRn5.

Lane 1: marker; lanes 2 to 4: whole-cell lysate, supernatant and pellet from uninduced cells at 23℃, respectively; lanes 5 to 7: whole-cell lysate, supernatant and pellet from induced cells at 23℃, respectively. lanes 8 to 10: whole-cell lysate, supernatant and pellet from uninduced cells at 37℃, respectively; lanes 11 to 13: whole-cell lysate, supernatant and pellet from induced cells at 37℃, respectively.

Then, we purified TRn5 by Immobilized Metal Affinity Chromatography (IMAC). However, the TRn5 expression level was too low to verify by SDS-PAGE (Fig. 3).

Fig. 3 SDS-PAGE of expression products of TRn5 purified by IMAC.

Lane 1: marker; lanes 2 to 11, induced cell sample at 23℃; lane 2: pellet; lane 3: sample washed with denaturing buffer with 8 mM urea; lane 4: sample after dialysis overnight; lane 5: sample after being bound to Ni-NTA resin; lane 6: sample eluted with 20 mM Tris-HCl; lane 7: sample eluted with 20 mM imidazole; lane 8: sample eluted with 50 mM imidazole; lane 9: sample eluted with 150 mM imidazole; lane 10: sample eluted with 300 mM imidazole; lane 11: sample eluted with 500 mM imidazole.

To optimize the TRn5 expression, we reviewed plenty of literature, from which we found that TRn5 could easily be dissolved in 5% acetic acid (pH=3) due to the existence of Histidine. Thus, we used a new protocol to obtain the purified TRn5. Solubilized in 5% acetic acid, the band of TRn5 was seen clearly, which means success of this purification manner (Fig. 4).

Fig. 5 SDS-PAGE of expression products of TRn5 using a new protocol.

Lane 1: marker; lanes 2 to 4: whole-cell lysate, supernatant and pellet from induced cells at 37℃, respectively; lane 5: sample washed with 5% acetic acid.

Protein self-healing

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Reference

[1] JUNG H, PENA-FRANCESCH A, SAADAT A, et al. Molecular tandem repeat strategy for elucidating mechanical properties of high-strength proteins[J]. PNAS, 2016, 113(23): 6478-6483.

[2] PENA-FRANCESCH A, JUNG H, DEMIREL M C, et al. Biosynthetic self-healing materials for soft machines [J]. Nat. Mater., 2020, 19(11): 1230-1235.

[3] PENA-FRANCESCH A, FLOREZ S, JUNG H, et al. Materials Fabrication from Native and Recombinant Thermoplastic Squid Proteins[J]. Adv. Funct., 2014, 24(47): 7401-7409.

[4] GUERETTE P A, HOON S, SEOW Y, et al. Accelerating the design of biomimetic materials by integrating RNA-seq with proteomics and materials science[J]. Nat. Biotechnol., 2013, 31(10): 908-915.

[5] DING D, GUERETTE P A, HOON S, et al. Biomimetic Production of Silk-Like Recombinant Squid Sucker Ring Teeth Proteins[J]. Biomacromolecules, 2014, 15(9): 3278-3289.