ISSN:
1365-2958
Source:
Blackwell Publishing Journal Backfiles 1879-2005
Topics:
Biology
,
Medicine
Notes:
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 (de-oxyribonuclease)/Nonidet extraction, treatment of the granules with guanidinium chloride followed by dialysis against buffer containing 0.5 M NaCI yielded a refolded, water-soluble PBP3**, which, upon chromatography on Superose 12, exhibited the expected 60000 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 PBP** can be obtained from 1 litre of culture of the overproducing strain.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1111/j.1365-2958.1988.tb00058.x
Permalink
