title: Efficient site-directed mutagenesis using an overlap extension-PCR method for expressing Mycoplasma hyopneumoniae genes in Escherichia coli.
abstract: Mycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia and results in significant economic losses in swine production worldwide. Vaccination is considered to be the most cost-effective strategy for control and prevention of this disease. However, the development of new recombinant subunit vaccines is often hampered by the unusual codon usage of this bacterium. To express M. hyopneumoniae proteins in heterologous systems such as Escherichia coli, the TGA codons that encode tryptophan in M. hyopneumoniae genes need to be replaced with the TGG codon. In this study we employed a modified overlap extension-PCR method for site-directed mutagenesis of selected TGA codons. Primers carrying the appropriate TGA to TGG mutation were employed in a two-step PCR amplification. The mutated PCR products were subsequently cloned into E. coli expression vectors. Using this method, we obtained 14 M. hyopneumoniae genes with up to three TGA to TGG substitutions per gene. Expression of the 10 mutated genes in E. coli was achieved. The method was rapid, simple and highly efficient in introducing the desired mutations in the A+T rich M. hyopneumoniae genes. In conclusion, this modified overlap extension-PCR method is suitable for large-scale site-directed mutagenesis of M. hyopneumoniae genes for heterologous expression.
authors.affiliation: Laboratório de Biologia Molecular, Centro de Biotecnologia, Universidade Federal de Pelotas, RS, Brazil.
authors.forename: Simone
authors.initials: S
authors.lastname: Simionatto
authors.forename: Silvana B
authors.initials: SB
authors.lastname: Marchioro
authors.forename: Vanessa
authors.initials: V
authors.lastname: Galli
authors.forename: Tessália D
authors.initials: TD
authors.lastname: Luerce
authors.forename: Daiane D
authors.initials: DD
authors.lastname: Hartwig
authors.forename: Angela N
authors.initials: AN
authors.lastname: Moreira
authors.forename: Odir A
authors.initials: OA
authors.lastname: Dellagostin
mesh_terms: D000942:Antigens, Bacterial / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D045705:Mycoplasma hyopneumoniae / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D000942:Antigens, Bacterial; D011994:Recombinant Proteins