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 recombinase 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). 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. 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 protein bands of the bacteria after attachment of the fusion tag as well as the original bacteria
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 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.
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) From Figure 8, after induced expression, the LSPET enzyme with the signal peptide added at the N-terminus was not directed into the fermentation broth. Reviewing the literature again we guessed that the recombinant target protein was most likely directed into the periplasmic space of E.coli. We therefore subsequently fragmented the fermentation broth after centrifugation of the fermentation broth and analyzed it again by SDS-PAGE.
Fig. 9 SDS-PAGE of the bacteria connected to the signal peptide as well as the induced expression proteins of the original bacteria (M: Marker; lane 1: 20 ℃ ISPET primordial cells broken supernatant; lane 2: 20 ℃ LSPET primordial cells broken precipitate solution; lane 3: 20 ℃ OmpA-LSPET cells broken supernatant; lane 4: 20 ℃ OmpA-LSPET cells broken precipitate solution; lane 5: 20 ℃ DsbA-LSPET cells broken precipitate solution). LSPET cells; lane 6: 20 ℃ DsbA-LSPET cells precipitation solution). 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. From Fig. 9, after induced expression, the soluble expression of LSPET enzyme protein after the addition of the signal peptide was greatly enhanced, and the comparison of the two signal peptides revealed that DsbA-LSPET significantly reduced the insoluble expression of LSPET enzyme in the precipitate, and the effect of OmpA-LSPET, although it also reduced the insoluble expression of LSPET enzyme in the precipitate, was not as obvious. The protein expression of OmpA-LSPET in the supernatant was significantly higher than that of LSPET primordia and DsbA-LSPET bacteria. It can be concluded that OmpA signal peptide can increase the soluble expression of LSPET enzyme by increasing the soluble expression of LSPET enzyme, but the effect is weaker than that of DsbA signal peptide in reducing the insoluble expression of LSPET enzyme. Both signal peptides were effective in the soluble expression of LSPET enzymes. 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 protein bands of the bacteria after linking the signal peptide as well as the original bacteria
molecular chaperones
Fig. 12 SDS-PAGE of the bacteria connected to molecular chaperones and of the induced expression proteins of the original bacteria(lane 1: primordial bacterium LSPET bacterial slime crushing solution supernatant; lane 2: primordial bacterium LSPET bacterial slime crushing solution precipitate; lane 3: connect molecular chaperone pTF16/LSPET bacterial slime crushing solution supernatant; lane 4: connect molecular chaperone pTF16/LSPET bacterial slime crushing solution precipitate; M: maker; lane 5: connect molecular chaperone pKJE7/LSPET bacterial slime breakage solution supernatant; lane 6: molecular chaperone pKJE7/LSPET bacterial slime breakage solution precipitate; lane 7: connect molecular chaperone pGro7/LSPET bacterial slime breakage solution supernatant; lane 6: molecular chaperone pGro7/LSPET bacterial slime breakage solution precipitate) From Figure 12, after induced expression, the soluble expression of LSPET enzyme after the addition of all three molecular chaperones was significantly enhanced, and the comparison of the three molecular chaperones revealed that pGro7/LSPET 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 are shown in Figures 13 and 14. Comparing pTF16/LSPET, pKJE7/LSPET, and pGro7/LSPET to LSPET, the soluble protein expression levels 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 gene protein expression.
Fig. 13 Histogram of the optical density analysis data of protein bands of the bacteria connected to the molecular chaperone as well as the original bacteria
Fig. 14 Percentage comparison of optical density analysis data of supernatant and precipitated protein bands of cell breakage solution from bacteria after linking molecular chaperones as well as the original bacteria In conclusion, the above three different strategies for enhancing soluble expression laid the foundation for the subsequent efficient soluble expression of PETase enzymes.
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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