Difference between revisions of "Part:BBa K4724083"

Revision as of 08:36, 12 October 2023

DsbA-LSPETase

Insert the signal peptide DsbA gene sequence into the vector pET-22b (+) - LSPET-12, and construct a recombinant plasmid using Gibson assembly method to promote the soluble expression of the original target fragment LSPET protein.

Characterize the results

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.

Sequence and Features