Difference between revisions of "Part:BBa K5101003"

Revision as of 10:05, 11 September 2024

Plasmid pETDuet-anti-TNF-α

Anti-TNF-α expression plasmid

Usage and Biology

Anti-TNF-α is a non-glycosylated monovalent Fab fragment of TNF-α monoclonal antibody with a molecular mass of 47.33 kDa. The Fab fragment is composed of a light chain (VL, CL domain) and a heavy chain (VH, CH1 domain) connected near the hinge region by disulfide bonds, with the light chain consisting of 214 amino acid residues and the heavy chain consisting of 229 amino acid residues. The pETDuet™-1 plasmid is designed for the co-expression of two target genes and contains two multiple cloning sites (MCS) for the co-expression of the two protein chains of Anti-TNF-α. Each MCS is preceded by a T7 promoter, lac operator, and a ribosome binding site (RBS). To ensure that Anti-TNF-α could be successfully assembled, we added ompA signal peptide sequence before the N-terminal end of the amino acid sequence of each chain. ompA signal peptide sequence can guide the transfer of secreted proteins to the secretory pathway for periplasmic translocation.

Construction of the plasmid

Construction of strain Nissle1917-pETDuet-anti-TNF-α

In the first multiple cloning site of the pETDuet vector, we selected the restriction enzyme sites Nco I and Sal I to clone and connect the ompA and light chain gene sequences onto the vector. For the second multiple cloning site, we chose the restriction enzyme sites Nde I and Xho I to connect the ompA and heavy chain genes to the vector. Through enzyme cutting and connection simulation using Snapgene software, we synthesized the corresponding plasmid pETDuet-anti-TNF-α, with the plasmid map shown in Figure 2-1-1.

Figure 2-1-1 Plasmid pETDuet-anti-TNF-α

As shown in Figure 2-1-2, the plasmid pETDuet-anti-TNFα is introduced into EcN through chemical transformation. After spreading the transformed cells on plates and incubating them at a constant temperature of 37°C for 12-16 hours, colonies containing the transformants can be obtained.

Figure 2-1-2 Strain Nissle 1917-pETDuet-anti-TNF-α Plate Image

The company's constructed plasmid DNA is transformed into EcN competent cells. Colonies with good growth on the transformation plates are picked and verified by colony PCR. The bacterial colonies with correct PCR results are activated and cultured. Plasmid pETDuet-anti-TNF-α is extracted from the validated bacterial strains and confirmed through plasmid PCR. According to Figure 2-1-3, the colony PCR results of single colonies Nissle 1917-pETDuet-anti-TNF-α-1, -2, and -3 show the target bands (The size of the target band is 2332 bp, which is a DNA fragment of certolizumab on plasmid pETDuet-anti-TNF-α), but with some non-specific bands, requiring further plasmid extraction for plasmid PCR verification. Lanes 1 to 3 represent single colonies Nissle 1917-pETDuet-anti-TNF-α -1, -2, and -3, using specific primers, while lane 4 serves as the PCR negative control.

Figure 2-1-3 Colony PCR gel electrophoresis of strain Nissle1917-pETDuet-anti-TNF-α

We performed plasmid PCR. Figure 2-1-4 shows that the plasmid PCR results of single colonies -1, -2, and -3 match the target bands, with a clean electrophoresis background, and the PCR negative control shows no bands. This indicates that the certolizumab’s Fab fragment gene has been successfully added to the expression vector pETDuet. The strain Nissle1917-pETDuet-anti-TNF-α construction is successful. Lanes 1 to 3 represent the plasmids of single colonies Nissle1917-pETDuet-anti-TNF-α-1, -2, and -3, while lane 4 is the PCR negative control.

Figure 2-1-3 Plasmid PCR gel electrophoresis of strain Nissle1917-pETDuet-anti-TNF-α

Construction of strain Nissle1917-pETDuet-anti-TNF-α-pKJE7

We sought help from NJTech-China, and they guided us on the relevant theory of protein misfolding and inclusion bodies. The pKJE7 plasmid can effectively express a group of molecular chaperones called the "Chaperone team," which cooperatively participate in protein folding. When the target protein is co-expressed with one of the "Chaperone team," it increases the recovery rate of soluble proteins and reduces the number of misfolded proteins due to inclusion body formation. We purchased the pKJE7 plasmid from Takara company and transferred it to the strain Nissle1917-pETDuet-anti-TNF-α, which was previously constructed, to construct the strain Nissle1917-pETDuet-anti-TNF-α-pKJE7. After picking colonies from several transformation plates of E. coli, plasmid extraction was performed, followed by PCR verification using specific primers. The target fragments were 2332bp and 460bp, where the 2332bp band corresponds to the certolizumab monoclonal antibody DNA fragment on the pETDuet-anti-TNF-α plasmid, and the 460bp band represents the chloramphenicol resistance DNA fragment on the molecular chaperone plasmid pKJE7. The results are shown in Figure 2-1-5. Lanes 1 to 3 represent single colonies of Nissle1917-pETDuet-anti-TNF-α-pKJE7-1, -2, and -3, while lanes 4 to 6 represent single colonies of Nissle1917-pETDuet-anti-TNF-α-pKJE7-1, -2, and -3, and lane 7 is the PCR negative control. The band positions are correct, indicating the successful construction of strain Nissle1917-pETDuet-anti-TNF-α-pKJE7.

Figure 2-1-5 Colony PCR gel electrophoresis of strain Nissle1917-pETDuet-anti-TNF-α-pKJE7

Protein expression validation

SDS-PAGE Verification

As shown in Figure 3-1-1 and 3-1-2,we expressed proteins in the strain Nissle1917-pETDuet-anti-TNF-α, purified and filtered after ultrasonic disruption, extracted the proteins, and used SDS-PAGE gel electrophoresis for protein gel verification. Since EcN is a probiotic bacterium, different from the commonly used E. coli BL21 (DE3) protein expression system, and lacks the modification processing system and chaperone function for eukaryotic proteins, endogenous proteases can degrade misfolded heterologous proteins. The Anti-TNF-α we expressed belongs to eukaryotic proteins and cannot be properly folded and expressed in the EcN strain.

As shown in Figure 3-1-1 and 3-1-2, we also expressed proteins in the strain Nissle1917-pETDuet-anti-TNF-α-pKJE7. After ultrasonic disruption, purification, and filtration, we extracted the proteins and used SDS-PAGE gel electrophoresis for protein gel verification. Theoretically, the pKJE7 plasmid can effectively express a group of molecular chaperones called the "Chaperone team," which cooperatively participate in protein folding. When the target protein is co-expressed with one of the "Chaperone team," it increases the recovery rate of soluble proteins and reduces the number of misfolded proteins due to inclusion body formation. However, we still did not achieve the desired bands, specifically the Anti-TNF-α segment (49.9KDa) containing the ompA signal peptide. Therefore, we will continue to explore in future experiments how to successfully synthesize and express Anti-TNF-α.

A: 1 - Supernatant after centrifugation of strain Nissle1917 culture; 3 - Supernatant after centrifugation of strain Nissle1917-anti-TNF-α culture; 4 - Supernatant after centrifugation of strain Nissle1917-anti-TNF-α-pKJE7 culture; 5 - Precipitate after centrifugation of strain Nissle1917 culture; 7 - Precipitate after centrifugation of strain Nissle1917-anti-TNF-α culture; 8 - Precipitate after centrifugation of strain Nissle1917-anti-TNF-α-pKJE7 culture

B: 1 - Supernatant after ultrasonication of strain Nissle culture; 3 - Supernatant after ultrasonication of strain Nissle-anti-TNF-α culture; 4 - Supernatant after ultrasonication of strain Nissle-anti-TNF-α-pKJE7 culture; 5 - Precipitate after ultrasonication of strain Nissle culture; 7 - Precipitate after ultrasonication of strain Nissle-anti-TNF-α culture; 8 - Precipitate after ultrasonication of strain Nissle-anti-TNF-α-pKJE7 culture

Figure 3-1-1 and Figure 3-1-2 SDS-PAGE results of the strain Nissle1917-pETDuet-anti-TNF-α and Nissle1917-pETDuet-anti-TNF-α-pKJE7

The molecular weight of the non-glycosylated Fab fragment of certolizumab has been confirmed to be 47.3 kDa. Since the certolizumab non-glycosylated Fab fragment in this study is a protein containing the ompA signaling peptide, the molecular weight of the certolizumab non-glycosylated Fab fragment from the recombinant strain Nissle 1917-pETDuet-anti-TNFα-pKJE7 is about the non-glycosylated Fab fragment of certolizumab with a molecular weight of about 49.9 kDa is clearly visible in the bands in lane 8 of Fig. 3-1-1 and 3-1-2. The SDS-PAGE results show that a recombinant EcN engineered strain with high efficiency in synthesizing the non-glycosylated Fab fragment of certolizumab was successfully constructed.

Through literature research, we have learned that the reducing cytoplasm of EcN is not conducive to the formation of disulfide bonds in Satumomab pendetide (Satumomab). While the signal peptide assists in folding the antibody in the periplasmic space, which is beneficial for disulfide bond formation, it still cannot achieve the high efficiency of disulfide bond expression as in strains like SHuffle T7-K12 containing disulfide bond isomerases. We plan to introduce a plasmid into EcN in the future that can express a disulfide bond isomerase to assist in the correct folding of Satumomab pendetide.