Overexpression, purification and biochemical characterization of a class A high-molecular-mass penicillin-binding protein (PBP), PBP1* and its soluble derivative from Mycobacterium tuberculosis.
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.
Bhakta
Sanjib
S
Department of Chemistry, Bose Institute, 93/1 Acharya Prafulla Chandra Road, Calcutta 700009, India.
Basu
Joyoti
J
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
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