Production and purification of the penicillin-binding protein 3 from Pseudomonas aeruginosa.
The penicillin-binding proteins (PBPs) are peripheral membrane enzymes that catalyze the final steps for the biosynthesis of the essential bacterial cell wall heteropolymer peptidoglycan. Bacteria produce a number and variety of PBPs which are classified as either high molecular weight or low molecular weight PBPs. The high molecular weight PBPs are multimodular being comprised of an N-terminal membrane anchor followed by a non-penicillin binding domain and a C-terminal penicillin-binding domain. The penicillin-binding domain functions as a serine-acyl transpeptidase to catalyze the crosslinking of neighboring glycan strands within the peptidoglycan sacculus. PBP 3 from Escherichia coli has been studied extensively and it has been shown to be responsible for the synthesis of peptidoglycan during the division and septation of the cells. The opportunistic human pathogen Pseudomonas aeruginosa produces a similar compliment of PBPs to E. coli, but differences in their organization and function have been noted. To investigate these differences further, appropriate quantities of each of the P. aeruginosa PBPs are required in forms amenable for study both in vivo and in vitro. Herein, we describe the cloning and expression of the ftsI gene encoding PBP 3from P. aeruginosa. The PBP was engineered in soluble form to facilitate its study in vitro and with a hexa-His tag to permit its facile purification by affinity chromatography. The recombinant proteins were demonstrated to bind penicillin and these forms of the PBPs were shown to be useful in studying their localization within their host cells by immunogold transmission electron microscopy.
de León
Sandra Ruiloba
SR
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1.
Daniels
Kathy
K
Clarke
Anthony J
AJ
D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005798:Genes, Bacterial; D006639:Histidine / Q000737:chemistry; D006801:Humans; D008958:Models, Molecular; D046915:Penicillin-Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D010406:Penicillins / Q000378:metabolism; D010457:Peptidoglycan / Q000096:biosynthesis / Q000378:metabolism; D010458:Peptidyl Transferases / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D011550:Pseudomonas aeruginosa / Q000378:metabolism* / Q000648:ultrastructure; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000648:ultrastructure; D012995:Solubility
D046915:Penicillin-Binding Proteins; D010406:Penicillins; D010457:Peptidoglycan; D011994:Recombinant Proteins; D006639:Histidine; D010458:Peptidyl Transferases