Part:BBa_K5101003:Design
Plasmid pETDuet-anti-TNF-α
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1560
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1560
Illegal NheI site found at 1766 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1560
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- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1560
Illegal AgeI site found at 1542 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
Gene Optimization and Synthesis: The genes encoding the light and heavy chains of the anti-TNF-α antibody, modeled after Certolizumab Pegol, were optimized for expression in E. coli. This optimization included codon usage adjustments to enhance translational efficiency in a bacterial system. Signal Peptide Usage: An ompA signal sequence was fused to each of the antibody chain genes. This signal sequence directs the nascent proteins to the periplasmic space of E. coli, which is crucial for the next step in protein maturation. The periplasmic location is advantageous for the formation of disulfide bonds, as it contains the oxidative environment and enzymes necessary for forming correct disulfide linkages, essential for the proper folding and functionality of the antibody. Disulfide Bond Formation: The natural oxidative environment of the E. coli periplasm promotes the formation of disulfide bonds between cysteine residues in the antibody chains. This step is critical for the structural integrity and biological activity of the anti-TNF-α antibody. Co-expression with Molecular Chaperones: After successful transformation of E. coli Nissle 1917 (EcN) with the pETDuet-anti-TNF-α plasmid, a second plasmid, pKJE7, was introduced. This plasmid encodes a set of molecular chaperones, which significantly enhance the correct folding of complex proteins. The co-expression of these molecular chaperones with the antibody chains helps to manage the proper folding of the protein, particularly in the challenging environment of the bacterial cell, reducing the formation of inactive misfolded proteins and inclusion bodies. Antibiotic Resistance and Selection: The plasmid includes antibiotic resistance genes that allow for the selection and maintenance of the plasmid in the bacterial culture, essential for scalable production. ===Source=== This plasmid, pETDuet-anti-TNF-α, is designed using the pETDuet-1 vector as its backbone to facilitate the co-expression of two proteins in E. coli. The vector contains two expression cassettes, each under the control of its own T7 lac promoter, allowing for simultaneous but independently controllable expression of the light and heavy chain genes of a non-glycosylated monovalent Fab fragment of the anti-TNF-α antibody, specifically modeled on Certolizumab Pegol. The genes encoding these antibody fragments were synthesized based on sequences adapted for bacterial expression and optimized for E. coli, including an ompA signal sequence to direct the proteins to the periplasm. This localization is critical for proper protein folding and disulfide bond formation, essential for the functionality of the antibody. Certolizumab Pegol, used as the model for these fragments, is known for its efficacy in neutralizing TNF-α without Fc-mediated functions, which can be advantageous in reducing immunogenicity. For validation and scientific comparison, similar sequences used in this design are accessible in the NCBI database, which provides extensive data on various therapeutic antibodies. ===References===