Study Of Codon Optimization, Detoxication Of Pseudomonas Aeruginosa Exotoxin A Gene And Its Characteristics

Pseudomonas aeruginosa exotoxin A (PEA) is a number of family of bacterial ADP-ribosylating toxins and an important causative agent of Pseudomonas aeruginosa. It can ADP-ribosylate the eukaryotic elongation factor 2 (EF-2) of target cells and inhibit protein synthesis, resulting in cytotoxicity to the host cells. Based on its cytotoxic function, specific targeting protein (antibody or ligand for membrane receptor) linked to cytotoxic PEA, called immunotoxins, were widely used for cancer therapy. In addition to cytotoxicity, PEA also has strong immunogenicity. It can be used not only as vaccine proteins, but also as vaccine adjuvants and vaccine carriers. However, due to its strong cytotoxicity, PEA can not be directly used for vaccine production as adjuvants and carriers. Only is PEA modified to lose its cytotoxic function by genetic engineering techniques, it can be used in vaccine production.To enhance PEA gene expression in E. coli and prepare detoxified PEA, we amplified PEA gene by PCR method from recombinant plasmid pcDNA3.1/PEA . According to this gene sequence, we optimized PEA gene codons using E.coli preferred codons. The optimized PEA gene was synthesized by a company. The wide-type and the codon-optimized PEA genes were inserted into pET20b(+) plasmid to construct His-tag-fused PEA prokaryotic expression vectors, pET20b-PEAwt/His and pET20b-PEAopt/His, respectively. Both vectors were transformed respectively into E. coli BL21 (DE3) pLysS and induced to express recombinant PEA by IPTG. The expressed PEA was released from periplasmic space of E. coli by osmotic shock with MgSO4 and purified by Ni-NTA agarose, then analyzed by SDS-PAGE and Western-blot. The concentration of PEA proteins were measured through Coomassie Protein Assay. Effect of codon optimization on gene expression in E. coli was analyzed by comparing both PEA gene expressions. For generation of detoxified PEA gene mutants, the several amino acid residues in PEA protein were chosen for substitution or deletion mutation based on PEA structural characteristics. The various mutants of PEA gene were generated by the method of nucleotide-mediated site-directed mutagenesis. Then, the various PEA gene mutants were transformed into E. coli BL21 (DE3) pLysS to express different PEA mutated proteins. Purification and identification for the different mutated PEA proteins were conducted with the same methods as above. The cytotoxicity of PEA mutants were detected using L929 mouse fibroblast cells. The low cytotoxic PEA mutants were screened from the different PEA mutants based on their cytotoxic effects on L929 cells.The results showed that after codon optimization, 402 bases have been substituted in PEA coding region, the GC content has been changed from 69.8 % in wide-type to 52.2 % in codon-optimized, resulting in a 33.7 % decrease. The PEA expression results showed that the expression level of codon optimized PEA gene (78.7±2.5 mg/L) was significantly higher than that of wild-type (39.0±2.0 mg/L) (P <0.01), increasing nearly 1-fold. The cytotoxicity test results displayed that PEA protein has obvious toxic effects on L929 cells, which indicated PEA protein possessed biological activity. SDS-PAGE results confirmed that all mutants of PEA could be expressed in E. coli in a secretory manner. The different mutated PEA proteins showed the different cytotoxic effects on L929 cells. Finally, the several low-cytotoxic PEA mutants were screened form all of the mutants according to their cytotoxic effects. As a result, 3 mutants having low cytotoxic effects were selected, which laid a foundation for further investigation on PEA as a vaccine adjuvants and carriers.