Part:BBa_K4724074
LSPETase
An enzyme that can degrade PET identified by our laboratory
Characterize the results
In order to enhance the solubility expression of LSPETase, we analyzed the reasons for the poor solubility expression of the LSPETase protein. We connected fusion tags, signal peptides, and molecular chaperones to the gene fragment, and modified the target gene to increase its solubility.
fusion tags
Fig. 3 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. 3, 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. 4 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. 5 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 4 and Figure 5 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.
signal peptides
Fig. 8 SDS-PAGE of recombinant protein expression products in the supernatant of fermentation broth under induction conditions of 20°C for 19h (M: Marker; lane 1: 20 ℃ supernatant of LSPET primordial cells crushed; lane 2: 20 ℃ precipitation solution of LSPET primordial cells crushed; lane 3: 20 ℃ supernatant of DsbA-LSPET fermentation broth; lane 4: 20 ℃ supernatant of OmpA-LSPET fermentation broth)
As depicted in Figure 8, it was observed that upon induction of expression, the LSPETase enzyme, when equipped with the N-terminal signal peptide, did not exhibit localization within the fermentation broth. Upon revisiting the relevant literature, it was hypothesized that the recombinant target protein was likely directed to the periplasmic compartment of E. coli. Consequently, to further investigate this phenomenon, the fermentation broth was subjected to centrifugation, followed by fragmentation, and subsequent analysis using SDS-PAGE.
Fig. 9 SDS-PAGE of the LSPETase and LSPETase fused with the signal peptide after induction at 20 ℃ for 19h (M: Marker; lane 1: Supernatant of LSPETase slurry; lane 2: Precipitate of LSPETase slurry; lane 3: Supernatant of OmpA-LSPETase slurry; lane 4: Precipitate of OmpA-LSPETase slurry; lane 5: Supernatant of DsbA-LSPETase slurry; lane 6: Precipitate of DsbA-LSPETase slurry.)
The target gene is known to express a protein length of 30.2 kDa, 32.3 kDa with the addition of the signal peptide DsbA and 32.2 kDa with the addition of the fusion tag OmpA.
As shown in Fig. 9, the soluble expression of LSPETase enzyme protein after the addition of the signal peptide was greatly enhanced. The comparison of the two signal peptides revealed that DsbA-LSPETase significantly reduced the insoluble expression of LSPETase enzyme in the precipitate, and the effect of OmpA-LSPETase, although it also reduced the insoluble expression of LSPETase enzyme in the precipitate, was not as obvious. The protein expression of OmpA-LSPETase in the supernatant was significantly higher than that of LSPETase and DsbA-LSPETase. It can be concluded that OmpA signal peptide can increase the soluble expression of LSPETase enzyme by increasing the soluble expression of LSPETase enzyme, but the effect is weaker than that of DsbA signal peptide in reducing the insoluble expression of LSPETase enzyme. Both signal peptides were effective in soluble expression of LSPETase.
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 analyzed as follows:
Fig. 10 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 signal peptides
Percentage comparison of optical density analysis data of the supernatant and precipitated protein bands of the bacterial cell breakage solution before and after the attachment of the signal peptides
An analysis of Figure 10 and Figure 11 showed that under the specified induction conditions, the protein solubility of DsbA-LSPETase and OmpA-LSPETase was increased by 1.7-fold and 1.4-fold respectively compared to the original LSPETase. The density values of the protein bands in the precipitate also decreased. This confirms the conclusion that the addition of signal peptides effectively facilitates the solubility of the target protein.
molecular chaperones
Fig. 12 SDS-PAGE of LSPETase, pTF16/LSPETase, pKJE7/LSPETase, and pGro7/LSPETase (M: maker; lane 1: Supernatant of LSPETase slurry; lane 2: Precipitate of LSPETase slurry; lane 3: Supernatant of pTF16/LSPETase slurry; lane 4: Precipitate of pTF16/LSPETase slurry; lane 5: Supernatant of pKJE7/LSPETase slurry; lane 6: Precipitate of pKJE7/LSPETase slurry; lane 7: Supernatant of pGro7/LSPETase slurry; lane 8: Precipitate of pGro7/LSPETase slurry.)
As depicted in Figure 12, the soluble expression of LSPETase enzyme after the addition of all three molecular chaperones was significantly enhanced, and the comparison of the three molecular chaperones revealed that pGro7/LSPETase was the most effective among them.
Further analysis of the protein gel was conducted to quantify the increase in protein expression using optical density measurements. The results were shown in Figure 13 and 14. Compared with LSPETase, the soluble protein expression levels of pTF16/LSPETase, pKJE7/LSPETase, and pGro7/LSPETase were increased by 2.3-fold, 1.8-fold, and 2.2-fold, respectively. The conclusion demonstrates that the addition of molecular chaperones significantly enhances the solubility of the target protein.
Fig. 13 Histogram of the optical density analysis data of supernatant and precipitated protein bands from the SDS-PAGE gel of LSPETase, pTF16/LSPETase, pKJE7/LSPETase, and pGro7/LSPETase
Fig. 14 Percentage comparison of optical density analysis data of supernatant and precipitated protein bands of cell breakage solution of LSPETase, pTF16/LSPETase, pKJE7/LSPETase, and pGro7/LSPETase
Conclusion
In conclusion, the above three different strategies for enhancing soluble expression laid the foundation for the subsequent efficient soluble expression of PETase enzymes.molecular chaperones of them has the best effect.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 832
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 88
Illegal AgeI site found at 175 - 1000COMPATIBLE WITH RFC[1000]
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