Article (Scientific journals)
Engineering and Overexpression of Periplasmic Forms of the Penicillin-Binding Protein 3 of Escherichia Coli
Fraipont, Claudine; Adam, Maggy; Nguyen-Distèche, Martine et al.
1994In Biochemical Journal, 298 (1), p. 189-195
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Keywords :
penicillin-binding protein 3; Escherichia coli; Truncated shape
Abstract :
[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.
Research center :
CIP - Centre d'Ingénierie des Protéines - ULiège
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Fraipont, Claudine ;  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-Distèche, 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
Language :
English
Title :
Engineering and Overexpression of Periplasmic Forms of the Penicillin-Binding Protein 3 of Escherichia Coli
Publication date :
10 February 1994
Journal title :
Biochemical Journal
ISSN :
0264-6021
eISSN :
1470-8728
Publisher :
Portland Press, London, United Kingdom
Volume :
298
Issue :
1
Pages :
189-195
Peer reviewed :
Peer Reviewed verified by ORBi
Available on ORBi :
since 13 May 2011

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