Cloning and expression of a transferrin-binding protein from Actinobacillus pleuropneumoniae.
An expression library was constructed from Actinobacillus pleuropneumoniae serotype 7. Escherichia coli transformants expressing recombinant proteins were identified by immunoscreening with porcine convalescent serum. One transformant expressing a 60-kDa protein (60K protein) in aggregated form was identified. Serum raised against the recombinant protein recognized a polypeptide with an indistinguishable electrophoretic mobility in the A. pleuropneumoniae wild type after iron-restricted growth only. The recombinant protein bound transferrin after blotting onto nitrocellulose. Using a competitive enzyme-linked immunosorbent assay (ELISA), the specificity of this binding for the amino-terminal half of iron-saturated porcine transferrin was established. Also, the 60K wild-type protein bound hemin as assessed by hemin-agarose chromatography. Hemin could inhibit transferrin binding of the recombinant protein in the competitive ELISA, whereas hemoglobin and synthetic iron chelators failed to do so. Southern blot analysis of several other A. pleuropneumoniae strains indicated that highly homologous sequence is present in eight of eight isolates of serotype 7 and in some isolates of serotypes 2, 3, and 4.
Gerlach
G F
GF
Veterinary Infectious Disease Organization, University of Saskatchewan, Saskatoon, Canada.
Anderson
C
C
Potter
A A
AA
Klashinsky
S
S
Willson
P J
PJ
D016977:Actinobacillus pleuropneumoniae / Q000737:chemistry* / Q000235:genetics; D000818:Animals; D002352:Carrier Proteins / Q000032:analysis / Q000235:genetics* / Q000502:physiology; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006418:Heme / Q000378:metabolism; D033862:Iron-Binding Proteins; D013552:Swine; D014168:Transferrin / Q000378:metabolism; D033863:Transferrin-Binding Proteins; D014169:Transformation, Bacterial
D002352:Carrier Proteins; D033862:Iron-Binding Proteins; D014168:Transferrin; D033863:Transferrin-Binding Proteins; D006418:Heme