Reference : Engineering and Overexpression of Periplasmic Forms of the Penicillin-Binding Protein...
Scientific journals : Article
Life sciences : Biochemistry, biophysics & molecular biology
http://hdl.handle.net/2268/90899
Engineering and Overexpression of Periplasmic Forms of the Penicillin-Binding Protein 3 of Escherichia Coli
English
Fraipont, Claudine mailto [Université de Liège - ULiège > Institut de Chimie > Centre d'Ingénierie des Protéines > > >]
Adam, Maggy [Université de Liège - ULiège > Institut de Chimie > Centre d'Ingénierie des Protéines > > >]
Nguyen-Disteche, Martine [Université de Liège - ULiège > Institut de Chimie > Centre d'Ingénierie des Protéines > > >]
Keck, Wolfgang [Rijksuniversiteit Groningen - RUG > Biochemie > Bioson Res Inst > >]
Van Beeumen, Jozef [Rijksuniversiteit-Gent > Vakgroep Biochemie, Fysiologie en Microbiologie > > >]
Ayala, Juan A. [Universidad Autonoma-Spain > C.S.I.C. > Centro de Biologio Molecular > >]
Granier, Benât [Université de Liège - ULiège > Institut de Chimie > Centre d'Ingénierie des Protéines > > >]
Hara, Hirashi [Misshima-Japan > National Institute of Genetics > > >]
Ghuysen, Jean-Marie [Université de Liège - ULiège > Institut de Chimie > Centre d'Ingénierie des Protéines > > >]
10-Feb-1994
Biochemical Journal
Portland Press
298
1
189-195
Yes (verified by ORBi)
International
0264-6021
1470-8728
London
United Kingdom
[en] penicillin-binding protein 3 ; Escherichia coli ; Truncated shape
[en] Replacement of the 36 and 56 N-terminal amino acid residues of the 588-amino-acid-residue membrane-bound penicillin-binding protein 3 (PBP3) of Escherichia coli by the OmpA signal peptide allows export of F37-V577 PBP3 and G57-V577 PBP3 respectively into the periplasm. The modified ftsI genes were placed under the control of the fused lpp promoter and lac promoter/operator; expression of the truncated PBP3s was optimized by varying the copy number of the recombinant plasmids and the amount of LacI repressor, and export was facilitated by increasing the SecB content of the producing strain. The periplasmic PBP3s (yield 8 mg/l of culture) were purified to 70% protein homogeneity. They require the presence of 0.25 M NaCl to remain soluble. Like the membrane-bound PBP3, they undergo processing by elimination of the C-terminal decapeptide I578-S588, they bind penicillin in a 1:1 molar ratio and they catalyse hydrolysis and aminolysis of acyclic thioesters that are analogues of penicillin. The membrane-anchor-free PBP3s have ragged N-termini. The G57-V577 PBP3, however, is less prone to proteolytic degradation than the F37-V577 PBP3.
Centre d'ingénierie des protéines - CIP
Researchers ; Professionals
http://hdl.handle.net/2268/90899

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