Difference between revisions of "Part:BBa K4724075"

Latest revision as of 08:33, 12 October 2023

NusA-LSPET

We fused the NusA fusion tag to the N-terminus of LSPETase to prevent self-aggregation and aggregation by binding and isolating its target protein's prone-to-aggregate folding intermediates, thereby ensuring stable expression of the recombinant protein.

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 BglII site found at 1368
Illegal XhoI site found at 2344
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 1600
Illegal AgeI site found at 1687
1000
COMPATIBLE WITH RFC[1000]

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-<p>Fig. 3 SDS-PAGE of recombinase with fusion tag attached
+<p>Fig. 1 SDS-PAGE of recombined LSPETase with fusion tag attached
-(M: Marker; Lane 1: Fusion tag NusA-conjugated bacterial slurry supernatant; Lane 2: Fusion tag NusA-conjugated bacterial slurry precipitated; Lane 3: Fusion tag TrxA-conjugated bacterial slurry precipitated; Lane 4: Fusion tag TrxA-conjugated bacterial slurry precipitated; Lane 5: Primordial LSPET bacterial slurry precipitated; Lane 6: Primordial LSPET bacterial slurry precipitated). LSPET slurry precipitate; lane 5: original LSPET slurry supernatant; lane 6: original LSPET slurry precipitate).
+(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>
-The target gene is known to express a protein length of 30.2 kDa, 87.0 kDa with the addition of the fusion tag NusA and 43.8 kDa with the addition of the fusion tag TrxA.
-From Fig. 3, after induced expression, the original bacteria (lanes 5 and 6) had less supernatant and more precipitation in the cell breakage solution at a length of 30.2 kDa; indicating that the soluble expression of the target protein was restricted. After the addition of fusion tag NusA (lanes 1 and 2), the colour of the 87.0kDa protein band (lane 1) in the supernatant of cell breakage solution was obviously deepened, and the colour of the 30.2kDa precipitated protein band (lane 2) was obviously lightened; after the addition of fusion tag TrxA (lanes 3 and 4), the colour of the 43.8kDa protein band (lane 3) in the supernatant of cell breakage solution was obviously deepened, and the colour of the 30.2kDa precipitated protein band was obviously lightened. After the addition of TrxA (lanes 3 and 4), the colour of the protein band with a length of 43.8 kDa (lane 3) in the supernatant of the cell breakage solution was obviously deepened, and the colour of the precipitated protein band with a length of 30.2 kDa (lane 4) was obviously lightened.
+<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>
-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>
+<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>
+<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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 <img src="https://static.igem.wiki/teams/4724/wiki/engineer4.png" style="height:40vh;">
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-<p>Fig. 4 Histogram of the optical density analysis data of protein bands of the bacteria after attachment of the fusion tag as well as the original bacteria</p>
+<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>
 <html>
 <img src="https://static.igem.wiki/teams/4724/wiki/engineer5.png" style="height:40vh;">
 </html>
-<p>Fig. 5 Percentage comparison of the optical density analysis data of the supernatant and precipitated protein bands of the cell breakage solution of the bacteria after attachment of the fusion tag as well as the original bacteria
+<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>
-An analysis of Figure 4 and Figure 5 shows that under the induction conditions, the protein solubility of TrxA-LSPET and NusA-LSPET, which are connected fusion tags, is significantly improved, with an increase of 3.1-fold and 1.7-fold respectively compared to LSPET. The density values of the protein bands in the precipitate also decreased. This further confirms the conclusion that the addition of fusion tags is effective in promoting the solubility of the target gene protein.</p>
+<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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