Open AccessExpression of Actinobacillus pleuropneumonia gene coding for Apx I protein in Escherichia coli
Author(s) -
Burdychova Radka,
Rychtera Milan,
Horvath Radek,
Dendis Milos,
Bartos Milan
Publication year - 2004
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1016/s0378-1097(03)00622-0
Subject(s) - actinobacillus pleuropneumoniae , pleuropneumonia , escherichia coli , biology , microbiology and biotechnology , fusion protein , expression vector , apx , actinobacillus , recombinant dna , gene , polymerase chain reaction , cloning (programming) , genetics , bacteria , biochemistry , peroxidase , programming language , computer science , enzyme
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.