Production and purification of the bacterial autolysin N-acetylmuramoyl-L-alanine amidase B from Pseudomonas aeruginosa.
The bacterial cell wall heteropolymer peptidoglycan is not a static structure as it is constantly being made and recycled throughout the bacterium's life cycle. This turnover of peptidoglycan is a highly coordinated event involving a complement of autolytic enzymes that include those with specificity for either the carbohydrate or the peptide linkages of peptidoglycan. One major class of these autolysins are the N-acetylmuramoyl-L-alanine amidases which cleave the amide linkage between the stem peptides and the lactyl moiety of muramoyl residues. They are required in the periplasm for cell separation during division and in both the periplasm and cytoplasm to trim soluble released PG fragments during turnover for recycling. The gene encoding N-acetylmuramoyl-L-alanine amidase B in Pseudomonas aeruginosa was cloned and over-expressed in Escherichia coli. The recombinant protein with a C-terminal His-tag was purified to apparent homogeneity by a combination of affinity and cation-exchange chromatographies using Ni(2+)NTA-agarose and Source S, respectively. Four separate assays involving zymography, light scattering, HPLC and MALDI-TOF mass spectrometry were used to confirm the activity of the protein as an N-acetylmuramoyl-L-alanine amidase.
Scheurwater
Edie M
EM
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada.
Pfeffer
John M
JM
Clarke
Anthony J
AJ
D000595:Amino Acid Sequence; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000378:metabolism; D008969:Molecular Sequence Data; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000096:biosynthesis* / Q000302:isolation & purification; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D009238:N-Acetylmuramoyl-L-alanine Amidase