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.

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

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

Characterization

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.

Protein expression

We expressed the protein in E.coli BL21 (DE3) using LB medium. After incubation at 37℃ for 5 h and 30℃ for 9 h, respectively, we found that most TRn5 (17.58 kDa) existed in precipitation as stated in previous research 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 denatured TRn5 with 8 mM urea and renatured it, which proved great protein losses as shown in SDS-PAGE. As a result, when we purified TRn5 by Immobilized Metal Affinity Chromatography (IMAC), the TRn5 expression level was too low to verify (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.

In order to optimize the expression of TRn5, we conducted a comprehensive review of the existing literature, revealing that the presence of Histidine facilitates the effortless dissolution of TRn5 in 5% acetic acid. Consequently, we implemented a novel protocol for the purification of TRn5. Upon solubilization in 5% acetic acid, a distinct and clear band of TRn5 was observed, thereby confirming the success of our purification approach (Figure 4).

Fig. 4 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

We obtained protein samples of TRn5 by freezedrying 24 h (Fig. 6).

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

Next, we dissolved protein samples in 5% acetic acid to reach 20 mg/μL, cast into square models and dried them at 70℃ for 3 h to obtain protein films. To examine the property of self-healing of TRn5, we punctured a TRn5 protein film to create a hole defect by a needle (Fig. 7a). After putting the punctured film at room temperature for 1 day, we clearly saw the hole defect healing (Fig. 7b).So it was proved that TRn5 has a self-healing property.

Fig. 7 Self-healing of TRn5 protein films after puncture damage. .

a. A hole defect was left by a needle through the film; b. Puncture damage was healed.

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.