Part:BBa_K5322032
Lpp-OmpA-Programmed Cell Death 1 Ligand 1 Functional Domain [Mus musculus] Expression System
Contents
Usage and Biology
During the project, we considered that PD-L1 might be encapsulated by mussel foot protein (Mfp) during expression and release, potentially reducing therapeutic efficacy. To address this, we utilized the surface display system Lpp-OmpA(BBa_K5322035) to present our passenger protein PD-L1 on the outer membrane of E. coli. This would facilitate its binding with PD-1, enhancing effectiveness. Additionally, to demonstrate surface display, we inserted a flexible protein linker and a TEV protease recognition site(BBa_K5322036) between Lpp-OmpA and the PD-L1 functional domain. By incubating the cells with TEV protease, PD-L1 could be cleaved from OmpA. Detection of the PD-L1 functional domain in the supernatant would indirectly confirm the success of surface display. We designed the plasmid pET29a-J23119-Lpp-OmpA-GISS-TEVsite-PD-L1(Functional domain)-T7.
Figure 1-1 Plasmid pET29a-J23119-Lpp-OmpA-GISS-TEVsite-PD-L1(Functional domain)-T7
Construction of the plasmid
The target fragment was amplified by PCR, and the pET29a vector was amplified. The PCR product was subjected to gel electrophoresis (30 min, 120 V). After the product was purified, homologous recombination was performed and transformed into Escherichia coli DH5α. The plate was inverted and cultured at 37°C. After 16 hours, colony PCR was performed on a single colony. The results are shown in the figure.
Figure 2-1 Colony PCR
The colony PCR was successfully inoculated at 37°C, 220rpm, 12-16h, the plasmid was extracted and sent to the company for sequencing. The sequencing results are shown in the figure below.
Figure 2-2 Sequencing results
According to the sequencing results, the plasmid construction was successful. The plasmid was transformed into EcN, the expression system was completed, and protein function testing was ready.
Protein characterization
The constructed system was tested for expression. After culturing in a 250 mL shake flask for 16 h, the cells were ultrasonically broken, passed through a nickel column, and the protein was purified. The samples were subjected to SDS-PAGE. The results are as follows.
Figure 3-1 SDS-PAGE
1: total protein; 2: supernatant; 3: precipitate; 4: after purification; 5: discarded after passing through nickel column; 6: before ultrafiltration; 7: after ultrafiltration;M: prestained protein marker
Protein gel verification cannot prove the expression of protein. For this reason, we consulted the literature and other means and speculated that the possible reason is that the amount of Lpp-OmpA-PD-L1 protein is still large and the bacterial expression is low. To solve this problem, we chose to use Western Blot. WB has higher sensitivity and can detect ng-level proteins. After a period of study, we mastered the operation and precautions of WB. We first directly performed WB experiments on total protein, and the results are shown in the figure below.
Figure 3-2 Western Blot
After verifying the presence of bands in the total protein, the correct expression of the target protein was confirmed. We then purified and ultrafiltered the protein and then directly performed WB verification. The results are shown in the figure below.
Figure 3-3 Western Blot
1: total protein; 2: supernatant; 3: precipitate; 4: after purification; 5: discarded after passing through nickel column; 6: before ultrafiltration; 7: after ultrafiltration;M: prestained protein marker
From the WB results, we can clearly see that due to the hydrophobicity of Lpp-OmpA, most of it exists in the precipitate, which is consistent with the theory. However, we can find that there are multiple bands in the band. After analysis, the possible reason is guessed: due to its characteristics, membrane proteins are destroyed when boiled at 100°C, resulting in protein breakage. Therefore, we designed an experiment based on the experience of predecessors to control the temperature of protein denaturation to explore the suitable temperature for membrane protein WB experiment. The results are as follows.
Figure 3-4 Western Blot
According to the WB result graph, we can find that under the deformation condition of 30℃ and 30min, the 34kDa protein is less damaged, so this denaturation condition is determined to be the preferred condition. So far, we have proved the expression of the target protein, and the next step is to prove the success of the surface membrane display system. 200mL of bacterial solution cultured for 12-16h and 50U TEV enzyme were incubated at 4℃ for 24h, and the supernatant was taken after centrifugation for ultrafiltration and concentration, and the bacteria were broken for standby. The two samples were subjected to the deformation condition of 30℃ and 30min for WB experimental verification. According to the WB results, PD-L1 (functional domain) exists in the incubation supernatant, which proves that the surface membrane display is successful.
Figure 3-5 Western Blot
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 832
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NheI site found at 790
Illegal NheI site found at 1415
Illegal PstI site found at 832 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 681
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 832
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 832
Illegal NgoMIV site found at 1221
Illegal AgeI site found at 1053 - 1000COMPATIBLE WITH RFC[1000]
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