Expression of Actinobacillus pleuropneumonia gene coding for Apx I protein in Escherichia coli.
This study presents cloning and expression of Actinobacillus pleuropneumoniae Apx I toxin in Escherichia coli expression system to produce fusion protein for the subsequent immunological studies. The gene coding Apx I toxin was amplified from the A. pleuropneumoniae serotype 10 DNA using polymerase chain reaction and cloned to vector under the control of strong, inducible T7 promoter. The presence of insert was confirmed by PCR screening and sequencing after the propagation of recombinant DNA in E. coli cells. The gene coding A. pleuropneumoniae Apx I toxin was extended with a segment to encode a polyhistidine tag linked to its C-terminal sequence allowing a one-step affinity purification of the complex with Ni-NTA resin. Expression of the Apx I coding sequence in E. coli resulted in the formation of insoluble inclusion bodies purified according to a standard purification protocol. The ease of this expression system, the powerful single-step purification and low costs make it possible to produce Apx I in large amounts to further study the role of Apx I in physiological processes.
Burdychova
Radka
R
Faculty of Chemistry, Brno University of Technology, Purkynova 118, 612 00 Brno, Czech Republic.
Rychtera
Milan
M
Horvath
Radek
R
Dendis
Milos
M
Bartos
Milan
M
D016977:Actinobacillus pleuropneumoniae / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001709:Biotechnology / Q000191:economics / Q000379:methods; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D006460:Hemolysin Proteins; D002479:Inclusion Bodies / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D006460:Hemolysin Proteins; D011994:Recombinant Proteins; C087744:ApxI toxin, Bacteria