Purification and characterization of a peptidoglycan-associated lipoprotein from Haemophilus influenzae.
We have purified to homogeneity a peptidoglycan-associated protein from Haemophilus influenzae. Our purification process used differential extraction of cell envelopes with nondenaturing detergents. Solubilization of this protein was accomplished by heating a peptidoglycan-enriched subcellular fraction in the presence of one of several nondenaturing detergents at 55-60 degrees C. The purified protein migrated as a single band, with a Mr approximately 15,000, following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein contains covalently linked fatty acids, is rich in tyrosine, but lacks methionine and tryptophan. Amino acid analysis also revealed the presence of glycerylcysteine, which has been shown to be the site of fatty acylation in other bacterial lipoproteins. Over 87% of the primary structure has been determined by sequencing high pressure liquid chromatography purified fragments derived from several endoproteinase digests. This protein belongs to a family of proteins, known as peptidoglycan associated lipoproteins, which appear to be components of the outer membranes of most Gram-negative bacteria.
Zlotnick
G W
GW
Department of Protein Chemistry, Praxis Biologics, Inc., Rochester, New York 14623.
Sanfilippo
V T
VT
Mattler
J A
JA
Kirkley
D H
DH
Boykins
R A
RA
Seid
R C
RC
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001425:Bacterial Outer Membrane Proteins; D002851:Chromatography, High Pressure Liquid; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000032:analysis; D029968:Escherichia coli Proteins; D005227:Fatty Acids / Q000032:analysis; D006193:Haemophilus influenzae / Q000032:analysis*; D006358:Hot Temperature; D008074:Lipoproteins / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010457:Peptidoglycan / Q000302:isolation & purification*; D011509:Proteoglycans; D012689:Sequence Homology, Nucleic Acid; D012995:Solubility
D000596:Amino Acids; D001425:Bacterial Outer Membrane Proteins; D003902:Detergents; D029968:Escherichia coli Proteins; C031163:ExcC protein, E coli; D005227:Fatty Acids; D008074:Lipoproteins; D010457:Peptidoglycan; D011509:Proteoglycans; C069988:PplA protein, Legionella pneumophila; D010450:Endopeptidases