Difference between revisions of "Part:BBa K4724074"

 
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<p>Fig. 3 SDS-PAGE of recombinase with fusion tag attached
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<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).
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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>
  
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.
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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>
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.
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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>
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>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. 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>
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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. 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
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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>
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>
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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>
  
 
<h3>signal peptides</h3>
 
<h3>signal peptides</h3>
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<p>Fig. 8 SDS-PAGE of recombinant protein expression products in the supernatant of fermentation broth under induction conditions of 20°C for 19h
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<p>Fig. 4 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)
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(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) </p>
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 <i>E.coli</i>.
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We therefore subsequently fragmented the fermentation broth after centrifugation of the fermentation broth and analyzed it again by SDS-PAGE.</p>
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<p>As depicted in Figure 4, 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 <i>E. coli</i>. Consequently, to further investigate this phenomenon, the fermentation broth was subjected to centrifugation, followed by fragmentation, and subsequent analysis using SDS-PAGE. </p>
  
 
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<p>Fig. 9 SDS-PAGE of the bacteria connected to the signal peptide as well as the induced expression proteins of the original bacteria
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<p>Fig. 5 SDS-PAGE of the LSPETase and LSPETase fused with the signal peptide after induction at 20 ℃ for 19h
(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).
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(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.) </p>
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.
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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.
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<p>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.</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 analyzed as follows:</p>
+
 
 +
<p>As shown in Fig. 5, 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. </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 analyzed as follows:</p>
  
 
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<p>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</p>
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<p>Fig. 6 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 </p>
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<p>Fig. 7 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</p>
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<p>An analysis of Figure 6 and Figure 7 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. </p>
  
 
<h3>molecular chaperones</h3>
 
<h3>molecular chaperones</h3>
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<p>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)
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<p>Fig. 8 SDS-PAGE of LSPETase, pTF16/LSPETase, pKJE7/LSPETase, and pGro7/LSPETase
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.
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(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.) </p>
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.
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</p>
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<p>As depicted in Figure 8, 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. </p>
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<p>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 9 and 10. 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. </p>
  
 
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<p>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</p>
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<p>Fig. 9 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</p>
  
 
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<p>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
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<p>Fig. 10 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</p>
In conclusion, the above three different strategies for enhancing soluble expression laid the foundation for the subsequent efficient soluble expression of PETase enzymes.</p>
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<h2 style="font-weight:600">Conclusion</h2>
 
<h2 style="font-weight:600">Conclusion</h2>
 
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<p>Among the above three experimental schemes to improve the solubility of LSPETase, the coupling of molecular chaperones has the best effect.</p>
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<p>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. </p>
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 08:31, 12 October 2023


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. 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.

signal peptides

Fig. 4 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 4, 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. 5 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. 5, 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. 6 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

Fig. 7 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 6 and Figure 7 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. 8 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 8, 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 9 and 10. 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. 9 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. 10 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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 832
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 88
    Illegal AgeI site found at 175
  • 1000
    COMPATIBLE WITH RFC[1000]