Difference between revisions of "Part:BBa K4724083"

Revision as of 20:00, 10 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. 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 analyszd 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

Fig. 11 Percentage comparison of optical density analysis data of the supernatant and precipitated protein bands of the cell shattering solution of the bacteria after linking the signal peptide as well as the original bacteria

An analysis of Figure 10 and Figure 11 shows that under the induction conditions, the protein solubility of DsbA-LSPET and OmpA-LSPET, which are connected signal peptides, is increased by 1.7-fold and 1.4-fold respectively compared to the original LSPET. The density values of the protein bands in the precipitate also decreased. This confirms the conclusion that the addition of signal peptides has a good effect in promoting the solubility of the target gene protein.

Sequence and Features