Escherichia coli; Bacteria; Enterobacteriaceae; Escherichieae; Characterization; Purification; Cytoplasm; Accumulation; Penicillin binding protein; N terminal-Sequence; Recombinant DNA; Secuencia N terminal; DNA recombinante; DNA recombinant; Bactérie; PBP3; Séquence N terminale; Cytoplasme; Protéine de liaison pénicilline; Citoplasma; Proteina de enlace penicilina
Abstract :
[en] Replacement of the amino-terminal 40-amino-acid region of the 588-amino-acid precursor of the membrane-bound penicillin-binding protein 3 (PBP3) by the decapeptide MKGKEFQAWI was carried out by altering the amino-coding end of the ftsI gene. Insertion of the modified gene into a runaway-replication plasmid under the control of a fused lpp 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 benzylpenicillin 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.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Bartholomé-De Belder, J.; Université de Liège - ULiège > Institut de Chimie > Service de Microbiologie
Nguyen-Distèche, Martine ; Université de Liège - ULiège > Institut de Chimie > Service de Microbiologie
Houba-Herin, N.; Université de Liège - ULiège > Institut de Chimie > Service de Microbiologie
Ghuysen, Jean-Marie ; Université de Liège - ULiège > Institut de Chimie > Service de Microbiologie
Maruyama, I. N.; National Institute of Genetics (Mishima - Japan) > Microbial Genetics
Hara, H.; National Institute of Genetics (Mishima - Japan) > Microbial Genetics
Hirota, Y.; National Institute of Genetics (Mishima - Japan) > Microbial Genetics
Inouye, M.; Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey > Department of Biochemistry
Language :
English
Title :
Overexpression, solubilization and refolding of a genetically engineered derivative of the penicillin-binding protein 3 of Escherichia coli K12.
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