Difference between revisions of "Part:BBa K5398005"

Revision as of 12:34, 20 September 2024

pET29a(+)-TRn5

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

Cloning strategy and results

In our project, we constructed pET29a(+)-TRn5 plasmid to express TRn5 by inserting the CDS of TRn5 into pET29a(+) vector by the following steps.(Fig. 1a).

• PCR amplification of TRn5 and pET29a(+) vector respectively. This PCR produces the pET29a(+)-TRn5 part ready for In-fusion Cloning (Fig. 1b and c).


Fig. 1 The plasmid map of pET29a(+)-TRn5 and 1% agarose gel electrophoresis of the PCR amplified pET29a(+)-TRn5 parts.

a. The plasmid map of pET29a(+)-TRn5; b. 1% agarose gel electrophoresis of the PCR amplified TRn5 (K5398001) (527 bp); c. 1% agarose gel electrophoresis of the PCR amplified pET29a(+) vector (5170 bp).

• In-fusion Cloning of purified PCR amplified TRn5 and the pET29a(+) vector parts for the efficient construction of the TRn5 coding sequence under the transcriptional control of the T7 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 pET29a(+)-TRn5 was conducted plasmid successfully.

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 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 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. 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 (Fig. 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.

Self-healing test

We obtained protein samples of TRn5 by freezedrying 24 h (Fig. 5). The final yield was about 15 mg/1 L bacterial culture. 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.



Fig. 5 The protein sample 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. 6a). After putting the punctured film at room temperature for 12 h, we clearly saw the hole defect healing (Fig. 6b).So it was proved that TRn5 has a self-healing property.



Fig. 6 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.