Characterization of the Pasteurella haemolytica transferrin receptor genes and the recombinant receptor proteins.
The tbpA and tbpB genes encoding the transferrin receptor proteins, TbpA and TbpB, from Pasteurella haemolytica A1 were cloned, sequenced and expressed in Escherichia coli. The genes were organized in a putative operon arrangement of tbpB- tbpA. The tbpB gene was preceded by putative promoter and regulatory sequences, and followed by a 96 base pair intergenic sequence in which no promoter regions were found, suggesting that the two genes are coordinately transcribed. The deduced amino acid sequences of the TbpA and TbpB proteins had regions of homology with the corresponding Neisseria meningitidis, N. gonorrhoeae, Haemophilus influenzae and Actinobacillus pleuropneumoniae Tbp and Lbp proteins. The intact tbpB gene was expressed in a T7 expression system and the resulting recombinant TbpB protein retained the functional bovine transferrin binding characteristics. The availability of the recombinant TbpB enabled us to demonstrate its specificity for ruminant transferrins, its ability to bind both the C-and N-terminal lobes of bovine transferrin and its preference for the iron-loaded form of this protein. Several attempts at expressing the cloned tbpA gene were unsuccessful, suggesting that the product of the gene may be toxic to E. coli.
Ogunnariwo
J A
JA
Department of Microbiology and Infectious Diseases, University of Calgary, Calgary, Alberta, T2N 4N1, Canada.
Woo
T K
TK
Lo
R Y
RY
Gonzalez
G C
GC
Schryvers
A B
AB
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D002417:Cattle; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006193:Haemophilus influenzae / Q000201:enzymology; D007438:Introns; D033862:Iron-Binding Proteins; D016978:Mannheimia haemolytica / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D009876:Operon; D011401:Promoter Regions, Genetic; D011990:Receptors, Transferrin / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D012418:Ruminants; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity; D014168:Transferrin / Q000378:metabolism; D033863:Transferrin-Binding Proteins
D002352:Carrier Proteins; D033862:Iron-Binding Proteins; D011990:Receptors, Transferrin; D011994:Recombinant Proteins; D014168:Transferrin; D033863:Transferrin-Binding Proteins