Expression, purification and characterization of recombinant phospholipase B from Moraxella bovis with anomalous electrophoretic behavior.
Moraxella bovis is the causative agent of infectious bovine keratoconjunctivitis (IBK) also known as pinkeye, a highly contagious and painful eye disease that is common in cattle throughout the world. Vaccination appears to be a reasonable and cost-effective means of control of pinkeye. Identification of genes encoding novel secreted antigens have been reported, and these antigens are being assessed for use in a vaccine. One of the genes encodes phospholipase B, which can be expressed with high purity and yield in recombinant Escherichia coli as a secreted, soluble, non-tagged, mature construct (less signal peptide with predicted mass 63 kDa). The recombinant phospholipase B exhibited anomalous electrophoretic mobility that was dependent on the temperature of the denaturing process, with bands observed at either 52 or 63 kDa. Analysis by in-gel digestion and liquid chromatography-mass spectrometry revealed these two distinct forms most likely had identical sequences. Phospholipase B is a compact, globular protein with a predicted structure typical of a conventional autotransporter. It is suggested that high temperature is required to unfold the protein (to denature the beta-barrel-rich transporter domain) and to ensure accessibility of the reducing agent. Interestingly, the two forms of the enzyme, differing in size and isoelectric points, were also detected in cell-free supernatants of M. bovis cultures, indicating that native phospholipase B may exist in two differentially folded states possibly also differing in oxidation status of cysteine residues.
Shiell
Brian J
BJ
Australian Animal Health Laboratory, CSIRO Livestock Industries, Geelong, Vic 3220, Australia.
Tachedjian
Mary
M
Bruce
Kerri
K
Beddome
Gary
G
Farn
Jacinta L
JL
Hoyne
Peter A
PA
Michalski
Wojtek P
WP
D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D019295:Computational Biology; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D008245:Lysophospholipase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D016955:Moraxella bovis / Q000201:enzymology*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D053719:Tandem Mass Spectrometry
D011994:Recombinant Proteins; D008245:Lysophospholipase