Part:BBa_K4724083
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 analysed 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 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 analysed as follows:
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1186
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 322
Illegal NgoMIV site found at 442
Illegal AgeI site found at 529 - 1000COMPATIBLE WITH RFC[1000]
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