Difference between revisions of "Part:BBa K5398005"
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<p>In order to obtain proteins with self-healing properties, we used  the pET29a(+) vector to express TRn5 (BBa_K5398001) (Fig. 1). We tried different strategies for TRn5 protein production and purification and tested its function. </p>
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<p>In order to obtain proteins with self-healing properties, we used  the pET29a(+) vector to express TRn5 (<a href="https://parts.igem.org/Part:BBa_K5398001"> BBa_K5398001)</a> ). We tried different strategies for TRn5 protein production and purification and tested its function. </p>
  
  
 
====Protein expression====
 
====Protein expression====
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<p>We expressed the protein in <i>E.coli</i> 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 and the TRn5 expression level at two temperatures had little difference (Fig. 1).</p>
 
<p>We expressed the protein in <i>E.coli</i> 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 and the TRn5 expression level at two temperatures had little difference (Fig. 1).</p>
  
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====Protein self-healing====
 
====Protein self-healing====

Revision as of 14:52, 17 September 2024


pET29a(+)-TRn5

In order to obtain proteins with self-healing properties, we used the pET29a(+) vector to express TRn5 (<a href="https://parts.igem.org/Part:BBa_K5398001"> BBa_K5398001)</a> ). 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 and the TRn5 expression level at two temperatures had little difference (Fig. 1).

Protein purification

Fig. 1 SDS-PAGE of expression products of TRn5.

Then, we purified TRn5 by Immobilized Metal Affinity Chromatography (IMAC). However, the TRn5 expression level was too low to verify by SDS-PAGE (Fig. 2).

模块示例

Protein purification

Fig. 2 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. 3).

模块示例

Protein purification

Fig. 3 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.