Overexpression, purification and biochemical characterization of a class A high-molecular-mass penicillin-binding protein (PBP), PBP1* and its soluble derivative from Mycobacterium tuberculosis
Bhakta, Sanjib and Basu, J. (2002) Overexpression, purification and biochemical characterization of a class A high-molecular-mass penicillin-binding protein (PBP), PBP1* and its soluble derivative from Mycobacterium tuberculosis. Biochemical Journal 361 (6), pp. 635-639. ISSN 0264-6021.
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. 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.
Metadata
| Item Type: | Article |
|---|---|
| School: | Birkbeck Faculties and Schools > Faculty of Science > School of Natural Sciences |
| Depositing User: | Sarah Hall |
| Date Deposited: | 30 Apr 2019 13:28 |
| Last Modified: | 02 Aug 2023 17:51 |
| URI: | https://eprints.bbk.ac.uk/id/eprint/27367 |
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