Purification and light-scattering analysis of penicillin-binding protein 4 from Escherichia coli.
Penicillin binding protein 4 (PBP4) from Escherichia coli is a protein involved in the recycling and maturation of the bacterial cell wall and it is inhibited by beta-lactam antibiotics. PBP4 exhibits D-Ala-D-Ala-endopeptidase as well as D-Ala-D-Ala-carboxypeptidase activity. To provide a structural template for the design of new, more specific antibiotics we started X-ray crystallographic studies of penicillin binding protein 4 from Escherichia coli. PBP4 has been overexpressed in Escherichia coli as a His-tagged protein. A large-sclae purification scheme, yielding a very pure material, has been set up and crystallization experiments have been started. Dynamic light scattering experiments suggested that PBP4 exhibits aggregation behavior with a number of different precipitating agents and additives. Only by addition of EDTA, PEG 4000, and ammonium sulfate is the molecular mass about 110 kDa.
Fusetti
F
F
Department of Chemistry, University of Groningen, The Netherlands.
Dijkstra
B W
BW
D001426:Bacterial Proteins; D002352:Carrier Proteins / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification*; D002614:Chelating Agents; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D029968:Escherichia coli Proteins; D006602:Hexosyltransferases; D008027:Light; D008970:Molecular Weight; D002267:Muramoylpentapeptide Carboxypeptidase / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification*; D046915:Penicillin-Binding Proteins; D010406:Penicillins / Q000378:metabolism*; D010458:Peptidyl Transferases; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012542:Scattering, Radiation; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase
D001426:Bacterial Proteins; D002352:Carrier Proteins; D002614:Chelating Agents; D029968:Escherichia coli Proteins; D046915:Penicillin-Binding Proteins; D010406:Penicillins; D011994:Recombinant Proteins; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase; C478706:penicillin-binding protein 4, E coli; D002267:Muramoylpentapeptide Carboxypeptidase