Reference : Overexpression, solubilization and refolding of a genetically engineered derivative o...
Scientific journals : Article
Life sciences : Biochemistry, biophysics & molecular biology
http://hdl.handle.net/2268/81283
Overexpression, solubilization and refolding of a genetically engineered derivative of the penicillin-binding protein 3 of Escherichia coli K12.
English
Bartholomé-De Belder, J. [Université de Liège - ULg > Institut de Chimie > Service de Microbiologie > >]
Nguyen-Distèche, Martine mailto [Université de Liège - ULg > Institut de Chimie > Service de Microbiologie > >]
Houba-Herin, N. [Université de Liège - ULg > Institut de Chimie > Service de Microbiologie > >]
Ghuysen, Jean-Marie [Université de Liège - ULg > 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 > > >]
1988
Molecular Microbiology
Blackwell Publishing
2
4
519-525
Yes (verified by ORBi)
International
0950-382X
1365-2958
Oxford
United Kingdom
[en] Escherichia coli ; Bacteria ; Enterobacteriaceae ; Escherichieae ; Characterization ; Purification ; Cytoplasm ; Accumulation ; Penicillin binding protein ; N terminal-Sequence ; Recombinant DNA ; Secuencia N terminal ; DNA recombinante
[fr] DNA recombinant ; Bactérie ; PBP3 ; Séquence N terminale ; Cytoplasme ; Protéine de liaison pénicilline ; Escherichia coli
[es] Escherichia coli ; Citoplasma ; Proteina de enlace penicilina
[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.
Researchers ; Professionals
http://hdl.handle.net/2268/81283
also: http://hdl.handle.net/2268/83879

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