title: Overexpression, purification and biochemical characterization of a class A high-molecular-mass penicillin-binding protein (PBP), PBP1* and its soluble derivative from Mycobacterium tuberculosis.
abstract: The product of the gene ponA present in cosmid MTCY21D4, one of the collection of clones representing the genome of Mycobacterium tuberculosis, has been named penicillin-binding protein 1* (PBP1*), by analogy to the previously characterized PBP1* of M. leprae. This gene has been overexpressed in Escherichia coli. His(6)-tagged PBP1* localizes to the membranes of induced E. coli cells. Its susceptibility to degradation upon proteinase K digestion of spheroplasts from E. coli expressing the protein supports the view that the majority of the protein translocates to the periplasmic side of the membrane. Recombinant PBP1* binds benzylpenicillin and several other beta-lactams, notably cefotaxime, with high affinity. Truncation of the N-terminal 64 amino acid residues results in an expressed protein present exclusively in inclusion bodies and unable to associate with the membrane. The C-terminal module encompassing amino acids 272-663 can be extracted from inclusion bodies under denaturing conditions using guanidine/HCl and refolded to give a protein fully competent in penicillin-binding. Deletion of Gly(95)-Gln(143) results in the expression of a protein, which is localized in the cytosol. The soluble derivative of PBP1* binds benzylpenicillin with the same efficiency as the full-length protein. This is the first report of a soluble derivative of a class A high-molecular-mass PBP.
authors.affiliation: Department of Chemistry, Bose Institute, 93/1 Acharya Prafulla Chandra Road, Calcutta 700009, India.
authors.forename: Sanjib
authors.initials: S
authors.lastname: Bhakta
authors.forename: Joyoti
authors.initials: J
authors.lastname: Basu
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins; D015153:Blotting, Western; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D019286:Endopeptidase K / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D017353:Gene Deletion; D005973:Glutamine / Q000737:chemistry; D005998:Glycine / Q000737:chemistry; D006602:Hexosyltransferases; D007700:Kinetics; D008969:Molecular Sequence Data; D002267:Muramoylpentapeptide Carboxypeptidase / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D009169:Mycobacterium tuberculosis / Q000378:metabolism*; D016366:Open Reading Frames; D046915:Penicillin-Binding Proteins; D010406:Penicillins / Q000494:pharmacology; D010458:Peptidyl Transferases; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D002352:Carrier Proteins; D046915:Penicillin-Binding Proteins; D010406:Penicillins; D011994:Recombinant Proteins; D005973:Glutamine; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase; D019286:Endopeptidase K; D005998:Glycine