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- Title
Overexpression, solubilization and refolding of a genetically engineered derivative of the penicillin-binding protein 3 of Escherichia coli K12.
- Authors
Bartholomé-De Belder, J.; Nguyen-Distèche, M.; Houba-Herin, N.; Ghuysen, J. M.; Maruyama, I. N.; Hara, H.; Hirota, Y.; Inouye, M.
- Abstract
Replacement of the amino-terminal 40-amino-acid region of the 588-ammo-acid precursor of the membrane-bound penicillin-binding protein 3 (P6P3) by the decapeptide MKGKEFQAWI was carried out by altering the amino-coding end of the ftsl gene. Insertion of the modified gene into a runaway-replication plasmid under the control of a fused Ipp promoter and lac promoter/operator, resulted in the overexpression by Escherichia coli of the modified PBP3 (designated PBP3**) in the cytoplasm. About 80% of the accumulated PBP3** underwent sequestration in the form of insoluble protein granules that were isolated by cell breakage or cell lysis. After selective removal of contaminants by an EDTA-lysozyme/DNase (deoxyribonuclease)/Nonidet extraction, treatment of the granules with guanidinium chloride followed by dialysis against buffer containing 0.5 M NaCl yielded a refolded, water-soluble PBP3**, which, upon chromatography on Superose 12, exhibited the expected 60 000 molecular mass. The refolded PBP3** bound benzytpenicillin in a 1 to 1 molar ratio, was highly sensitive to aztreonam and showed the same degree of thermostability, in terms of penicillin-binding capacity, as the parent, membrane-bound PBP3, suggesting that protein refolding occurred with formation of the correct intramolecular interactions. Two to three mg of refolded PBP3** can be obtained from 1 litre of culture of the overproducing strain.
- Subjects
GENETIC engineering; PENICILLIN; CARRIER proteins; ESCHERICHIA coli; GENETIC recombination; BIOLOGICAL transport; PROTEIN binding; BACTERIAL genetics; GENETICS
- Publication
Molecular Microbiology, 1988, Vol 2, Issue 4, p519
- ISSN
0950-382X
- Publication type
Article
- DOI
10.1111/j.1365-2958.1988.tb00058.x