Part:BBa_K5398001

TRn5

Fig. 2 Protein experiment experiment of SUMO-TRn4-mfp5(35.4 KDa).

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

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) (Pena-Francesch et al., 2020), 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.

In order to obtain proteins with self-healing properties, we used the pET29a(+) vector to express TRn5 (BBa_K5398001) (Fig. 2). We tried different strategies for TRn5 protein production and purification and tested its function.

Fig. 2 The plasmid map of pET29a-TRn5.

We expressed the protein in E.coli BL21 (DE3) using LB medium. After incubation at 37℃ for 5h and 30℃ for 9h, respectively, we found that most TRn5 (17.58 kDa) existed in precipitation and the TRn5 expression level at two temperatures had little difference (Fig. 3).

Fig. 3 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. 4).

Fig. 4 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. 5).

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.

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.

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]