A Single Codon Optimization Enhances Recombinant Human TNF- Vaccine Expression in .

As a proinflammatory cytokine, tumor necrosis factor-alpha (TNF-) plays a pivotal role in various autoimmune diseases such as rheumatoid arthritis (RA). Thus, TNF- has been defined as a therapeutic target for RA. Although some TNF- antagonists including neutralizing monoclonal antibodies and soluble receptors have been approved to be successful in attenuating symptoms in patients suffering from RA, the long-term use of these passive immunization reagents could cause some problems like a variable degree of immunogenicity. In the present study, in order to wake up active immune responses of RA patients, we developed a recombinant TNF- therapeutic vaccine (named mrTNF-PADRE) by coupling a 12-amino acid universal Pan HLA-DR Epitope (PADRE) to the protein. Codon optimization was performed to improve the secondary structure of mrTNF-PADRE mRNA to ensure its heterologous expression. As a result, a single codon synonymous mutation greatly elevated recombinant protein expression (about 30% of the total bacteria proteins) in as compared with the undetectable expression of the unoptimized gene. Although expressed as insoluble inclusion bodies (IBs), the vaccine can be effectively prepared with a purity of over 95% by IBs washing and one-step gel-infiltration chromatography. By this strategy, a stable yield of 5.2 mg purified mrTNF-PADRE per gram of cell paste could be obtained.