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. His6-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 β-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 Gly95—Gln143 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.
- cell wall biosynthesis
Abbreviations used: CTAB, cetyltrimethylammonium bromide; IPTG, isopropyl β-d-thiogalactoside; NTA, nitrilotriacetate; ORF, open reading frame; PB, penicillin-binding; PBP, PB protein; n-PB module, non-PB module; PB-PBP1∗, truncated PBP1∗ lacking residues Val2—Gln271; s-PBP1∗, soluble PBP1∗ (truncated PBP1∗ lacking residues Gly95—Gln143); Zwittergent 3–12, N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulphonate.
- The Biochemical Society, London ©2002