title: Molecular cloning, partial purification, and characterization of a haemin-binding lipoprotein from Haemophilus influenzae type b.
abstract: A library of genomic DNA fragments from Haemophilus influenzae type b (Hib) DL42 was constructed in plasmid pBR322, transformed into Escherichia coli strain RR1, and screened for recombinant clones with haemin-binding activity by plating onto haemin-containing agar. Expression of haemin-binding activity by clones correlated with the expression of a protein with an apparent molecular weight of 51,000 (51K) that was also recognized by anti-Hib strain DL42 serum in immunoblots. One recombinant clone, designated pHM2, with the smallest DNA insert (3.62 kb) was characterized further. Ethanol inhibition of expression of pHM2 in minicells revealed that the 51K protein was the result of a processing event involving a larger precursor. E. coli RR1(pHM2) adsorbed haemin in liquid suspensions as well as from solid media. Subcloning of a 2.6 kb fragment of pHM2 into a shuttle vector permitted the construction of a recombinant Hib clone, DL42(pHM1002), which overexpressed the 51K haemin-binding protein. This 51K protein appears to be peripherally associated with the inner, and possibly outer, membranes of Hib. Affinity chromatography on haemin-agarose was utilized to purify the haemin-binding protein from both E. coli RR1(pHM2) and Hib DL42(pHM1002) to near homogeneity. The use of the antibiotic globomycin in a minicell expression system and radioimmunoprecipitation analysis of Hib proteins intrinsically radiolabelled with [3H]-palmitate indicated that the 51K haemin-binding protein is a lipoprotein.
authors.affiliation: Department of Microbiology, University of Texas, Southwestern Medical Center, Dallas 75235.
authors.forename: M S
authors.initials: MS
authors.lastname: Hanson
authors.forename: E J
authors.initials: EJ
authors.lastname: Hansen
mesh_terms: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins / Q000235:genetics*; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002458:Cell Fractionation; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D000431:Ethanol / Q000494:pharmacology; D005798:Genes, Bacterial; D015698:Genomic Library; D006193:Haemophilus influenzae / Q000235:genetics*; D000081062:Heme-Binding Proteins; D006420:Hemeproteins / Q000235:genetics*; D006427:Hemin / Q000378:metabolism*; D015151:Immunoblotting; D008074:Lipoproteins / Q000235:genetics / Q000378:metabolism; D008970:Molecular Weight; D016254:Mutagenesis, Insertional; D010455:Peptides / Q000494:pharmacology; D015531:Radioimmunoprecipitation Assay; D015183:Restriction Mapping
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D002352:Carrier Proteins; D000081062:Heme-Binding Proteins; D006420:Hemeproteins; D008074:Lipoproteins; D010455:Peptides; D000431:Ethanol; C017339:globomycin; D006427:Hemin