Difference between revisions of "Part:BBa K4724085"

 
 
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We fused the NusA tag to the N-terminus of LSPETase to enhance its folding characteristics and increase the solubility of the recombinant protein, thereby reducing the formation of inclusion bodies.
 
We fused the NusA tag to the N-terminus of LSPETase to enhance its folding characteristics and increase the solubility of the recombinant protein, thereby reducing the formation of inclusion bodies.
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<h2 style="font-weight:600">Characterize the results</h2>
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<p>Fig. 1 SDS-PAGE of recombined LSPETase with fusion tag attached
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(M: Marker; Lane 1: Supernatant of NusA-LSPETase slurry; Lane 2: Precipitate of NusA-LSPETase slurry; Lane 3: Supernatant of TrxA-LSPETase slurry; Lane 4: Precipitate of TrxA-LSPETase slurry; lane 5: Supernatant of original LSPETase slurry; lane 6: Precipitate of original LSPETase slurry) </p>
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<p>TThe molecular weight of LSPETase is 30.2 kDa, while NusA-LSPETase and TrxA-LSPETase are 87.0 kDa and 43.8 kDa, respectively. </p>
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<p>As depicted in Fig. 1, original LSPETase (lanes 5 and 6) had less supernatant and more precipitation in the cell breakage solution, indicating that the soluble expression of the target protein was restricted. After the addition of fusion tag NusA (lanes 1 and 2), the concentration of the 87.0 kDa protein band (lane 1) in the supernatant of cell breakage solution was obviously improved, and the precipitated protein band (lane 2) was obviously lightened. And after the addition of fusion tag TrxA (lanes 3 and 4), the concentration of the 43.8 kDa protein band (lane 3) in the supernatant of cell breakage solution was obviously improved, and the precipitated protein band was obviously lightened. </p>
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<p>Further optical density analysis was performed on protein gels at induction conditions of 20°C for 19 h to quantify the increase in protein expression, and the results were analysed as follows:</p>
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<p>Fig. 2 Histogram of the optical density analysis data of supernatant and precipitated protein bands from the SDS-PAGE gel of the bacterial cell breakage solution before and after the attachment of the fusion tag</p>
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<p>Fig. 3 Percentage comparison of the optical density analysis data of the supernatant and precipitated protein bands of the bacterial cell breakage solution before and after the attachment of the fusion tag</p>
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<p>An examination of Figure 2 and Figure 3 revealed that, under the specified induction conditions, the protein solubility of TrxA-LSPETase and NusA-LSPETase, exhibited a remarkable enhancement, with a respective increase of 3.1-fold and 1.7-fold compared to LSPETase. Moreover, the density values of the protein bands in the precipitate exhibited a reduction. This provides additional validation for the inference that the incorporation of fusion tags effectively facilitates the solubility of the target protein. </p>
  
 
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Latest revision as of 08:34, 12 October 2023


TrxA-LSPETase


We fused the NusA tag to the N-terminus of LSPETase to enhance its folding characteristics and increase the solubility of the recombinant protein, thereby reducing the formation of inclusion bodies.

Characterize the results

Fig. 1 SDS-PAGE of recombined LSPETase with fusion tag attached (M: Marker; Lane 1: Supernatant of NusA-LSPETase slurry; Lane 2: Precipitate of NusA-LSPETase slurry; Lane 3: Supernatant of TrxA-LSPETase slurry; Lane 4: Precipitate of TrxA-LSPETase slurry; lane 5: Supernatant of original LSPETase slurry; lane 6: Precipitate of original LSPETase slurry)

TThe molecular weight of LSPETase is 30.2 kDa, while NusA-LSPETase and TrxA-LSPETase are 87.0 kDa and 43.8 kDa, respectively.

As depicted in Fig. 1, original LSPETase (lanes 5 and 6) had less supernatant and more precipitation in the cell breakage solution, indicating that the soluble expression of the target protein was restricted. After the addition of fusion tag NusA (lanes 1 and 2), the concentration of the 87.0 kDa protein band (lane 1) in the supernatant of cell breakage solution was obviously improved, and the precipitated protein band (lane 2) was obviously lightened. And after the addition of fusion tag TrxA (lanes 3 and 4), the concentration of the 43.8 kDa protein band (lane 3) in the supernatant of cell breakage solution was obviously improved, and the precipitated protein band was obviously lightened.

Further optical density analysis was performed on protein gels at induction conditions of 20°C for 19 h to quantify the increase in protein expression, and the results were analysed as follows:

Fig. 2 Histogram of the optical density analysis data of supernatant and precipitated protein bands from the SDS-PAGE gel of the bacterial cell breakage solution before and after the attachment of the fusion tag

Fig. 3 Percentage comparison of the optical density analysis data of the supernatant and precipitated protein bands of the bacterial cell breakage solution before and after the attachment of the fusion tag

An examination of Figure 2 and Figure 3 revealed that, under the specified induction conditions, the protein solubility of TrxA-LSPETase and NusA-LSPETase, exhibited a remarkable enhancement, with a respective increase of 3.1-fold and 1.7-fold compared to LSPETase. Moreover, the density values of the protein bands in the precipitate exhibited a reduction. This provides additional validation for the inference that the incorporation of fusion tags effectively facilitates the solubility of the target protein.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 1192
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 322
    Illegal NgoMIV site found at 448
    Illegal AgeI site found at 535
  • 1000
    COMPATIBLE WITH RFC[1000]