Part:BBa_K5398001:Experience

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Applications of BBa_K5398001

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

Cloning strategy and results

In our project, we first synthesized pET-11b-TRn5 plasmid and attemped to express it in E.coli BL21 (DE3) using LB medium. However, it didn't express our targeting protein TRn5. So, to continue our project, we constructed pET-29a(+)-TRn5 plasmid to express TRn5 (Fig. 1a).

• PCR amplification of TRn5 and pET-29a(+) vector respectively. This PCR produced the pET-29a(+)-TRn5 part ready for In-fusion Cloning (Fig. 1b,c).


Fig. 1 | The plasmid map of pET-29a(+)-TRn5 and 1% agarose gel electrophoresis of the PCR amplified pET-29a(+)-TRn5 parts.

a. The plasmid map of pET-29a(+)-TRn5. b. 1% agarose gel electrophoresis of the PCR amplified TRn5 (K5398001) (527 bp). c. 1% agarose gel electrophoresis of the PCR amplified pET-29a(+) vector (5170 bp).

• In-fusion Cloning of purified PCR amplified TRn5 and the pET-29a(+) vector parts for the efficient construction of the TRn5 coding sequence under the transcriptional control of the T7lac promoter. The recombinant plasmid was transferred into E.coli DH5α.
• Verification of target recombinant plamid. Colony PCR was used to screen for clones with inserts of the desired sizes and Sanger sequencing confirmed the lengths and compositions of the clones after plasmid isolation, from which we concluded that the pET-29a(+)-TRn5 was conducted plasmid successfully (Fig. 2).


Fig. 2 | Verification of recombinant plamid pET-29a(+)-TRn5.

a. 1% agarose gel electrophoresis of colony PCR of using T7 and T7 ter primers. b. The result of sequencing the TRn5 of the recombinant plasmid.

Protein expression

We expressed the protein in E.coli BL21 (DE3) using LB medium. After incubation at 23℃ for 16 h and 37℃ for 5 h respectively, we found that most TRn5 (17.58 kDa) existed in precipitate as stated in previous research 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 2-4: whole-cell lysate, supernatant and pellet from uninduced cells at 23℃, respectively; Lanes 5-7: whole-cell lysate, supernatant and pellet from induced cells at 23℃, respectively; Lanes 8-10: whole-cell lysate, supernatant and pellet from uninduced cells at 37℃, respectively; Lanes 11-13: whole-cell lysate, supernatant and pellet from induced cells at 37℃, respectively.

Then, we denatured TRn5 with 8 M urea overnight and renatured it by dialysis, 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. 4).



Fig. 4 | SDS-PAGE of expression products of TRn5 purified by IMAC.

Lane 1: marker; Lanes 2-11, induced cell samples at 23℃; Lane 2: pellet; Lane 3: sample washed with denaturing buffer with 8 M 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 7-11: samples eluted with 20, 50, 150, 300 and 500 mM imidazoles.

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 (Fig. 5).



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

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

Self-healing test

We obtained protein samples of TRn5 by freezedrying 24 h (Fig. 6). The final yield was about 150.4 mg/L bacterial culture. Next, we dissolved protein samples in 5% acetic acid to reach 20 mg/μL, cast them into square models and dried them at 70℃ for 3 h to obtain protein films.



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

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 12 h, 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.

More information about the project for which the part was created: SAMUS (NAU-CHINA 2024).

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.

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