title: Codon optimization enhances secretory expression of Pseudomonas aeruginosa exotoxin A in E. coli.
abstract: Pseudomonas aeruginosa exotoxin A (PEA) is a number of family of bacterial ADP-ribosylating toxins and possesses strong immunogenicity. The detoxified exotoxin A, as a potent vaccine adjuvant and vaccine carrier protein, has been extensively used in human and animal vaccinations. However, the expression level of PEA gene in Escherichia coli is relative low which is likely due to the presence of rare codon and high levels of GC content. In order to enhance PEA gene expression, we optimized PEA gene using E. coli preferred codons and expressed it in E. coli BL21 (DE3) by using pET-20b(+) secretory expression vector. Our results showed that codon optimization significantly reduced GC content and enhanced PEA gene expression (70% increase compared with that of the wild-type). Moreover, the codon-optimized PEA possessed biological activity and had the similar toxic effects on mouse L292 cells compared with the wild-type PEA gene. Codon optimization will not only improve PEA gene expression but also benefit further modification of PEA gene using nucleotide-mediated site-directed mutagenesis. A large number of purified PEA proteins will provide the necessary conditions for further PEA functional research and application.
authors.affiliation: Department of Basic Veterinary Medicine, College of Animal Science and Veterinary Medicine, Agricultural University of Hebei, Baoding 071001, China.
authors.forename: Xingxing
authors.initials: X
authors.lastname: Wang
authors.forename: Xiujin
authors.initials: X
authors.lastname: Li
authors.forename: Zhenlong
authors.initials: Z
authors.lastname: Zhang
authors.forename: Xinliang
authors.initials: X
authors.lastname: Shen
authors.forename: Fei
authors.initials: F
authors.lastname: Zhong
mesh_terms: D036002:ADP Ribose Transferases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival; D003001:Cloning, Molecular / Q000379:methods*; D003062:Codon / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005098:Exotoxins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D011550:Pseudomonas aeruginosa / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D037521:Virulence Factors / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D003062:Codon; D005098:Exotoxins; D011994:Recombinant Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A