Identification and sequence determination of recombinant Clostridium perfringens alpha-toxin by use of electrospray ionization mass spectrometry.
Only a few methods exist for simple, sensitive and rapid detection of alpha-toxin in clinical and biological samples. The aim of our study was to establish a procedure for the production of an antibody against a recombinant antigen with confirmed sequence identity. We applied a noble approach based on proteomics using a mass spectrometer for the conclusive identification of the recombinant alpha-toxin that was subsequently used as an antigen. The recombinant alpha-toxin was produced in Escherichia coli. A clinical isolate of Clostridium perfringens GAI 94074 was amplified by polymerase chain reaction (PCR) and subsequently, cloning was performed. Three different fragments were cloned using a pET100/D-TOPO vector. These fragments coded for a ribosome binding site, a signal peptide and the alpha-toxin gene, respectively. Recombinant pET100 plasmids were cloned into TOP 10 cells and the isolated plasmids were transferred into BL21 Star (DE3) cells. Their expression was then induced with isopropyl-beta-D-thiogalactopyranoside (IPTG). Recombinant E. coli transformed with a plasmid encoding the alpha-toxin gene alone produced a biologically inactive protein. On the other hand, E. coli carrying the plasmid encoding the toxin sequence and its native signal peptide sequence, or the toxin sequence along with the ribosome binding sequence and the signal peptide sequence secreted an active alpha-toxin with phospholipase activity. Accordingly, the C. perfringens gene encoding the alpha-toxin protein along with its signal peptide was successfully cloned, expressed, and secreted by E. coli. Furthermore, without consideration of its activity, we used mass spectrometry to confirm that the expressed protein was indeed the alpha-toxin. Thus, the identification of alpha-toxin protein using both the biological activity testing and the mass spectrometry analysis is expected to verify the significant production of C. perfringens antibody. The study for the analysis of recombinant alpha-toxin using ESI/MS has not been reported. In this study, we report the successful cloning, expression, secretion, identification and sequence determination of the C. perfringens alpha-toxin.
Advanced Research Center for Life Science and Human, Department of Applied Molecular Chemistry, Graduate School of Industrial Technology, Nihon University, 1-2-1 Izumicho, Narashino, Chiba 275-8575, Japan.
Hitoshi
H
Saito
Masaharu
M
Inoue
Masayoshi
M
Tomiki
Hiroshi
H
Nemoto
Tomoe
T
Komoriya
Junko
J
Kimata
Kunitomo
K
Watanabe
Hideki
H
Kohno
D001427:Bacterial Toxins / Q000276:immunology / Q000302:isolation & purification*; D002135:Calcium-Binding Proteins / Q000276:immunology / Q000302:isolation & purification*; D019976:Cloning, Organism; D003016:Clostridium perfringens; D004926:Escherichia coli; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000302:isolation & purification; D021241:Spectrometry, Mass, Electrospray Ionization; D010738:Type C Phospholipases / Q000276:immunology / Q000302:isolation & purification*
D001427:Bacterial Toxins; D002135:Calcium-Binding Proteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D010738:Type C Phospholipases; C040649:alpha toxin, Clostridium perfringens