Expression, purification and characterization of recombinant phospholipase B from Moraxella bovis with anomalous electrophoretic behavior
| Autor: | Wojtek P. Michalski, Peter A. Hoyne, Jacinta L. Farn, Mary Tachedjian, Kerri Bruce, Brian J. Shiell, Gary Beddome |
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| Rok vydání: | 2007 |
| Předmět: |
Signal peptide
Moraxella bovis law.invention Antigen law Tandem Mass Spectrometry Electrophoresis Gel Two-Dimensional Cloning Molecular Chromatography High Pressure Liquid chemistry.chemical_classification Phospholipase B biology Computational Biology biology.organism_classification Molecular biology Recombinant Proteins Enzyme chemistry Biochemistry Infectious bovine keratoconjunctivitis Recombinant DNA Electrophoresis Polyacrylamide Gel Lysophospholipase Biotechnology Cysteine |
| Zdroj: | Protein expression and purification. 55(2) |
| ISSN: | 1046-5928 |
| Popis: | Moraxella bovis is the causative agent of infectious bovine keratoconjunctivitis (IBK) also known as pinkeye, a highly contagious andpainful eye disease that is common in cattle throughout the world. Vaccination appears to be a reasonable and cost-effective means ofcontrol of pinkeye. Identification of genes encoding novel secreted antigens have been reported, and these antigens are being assessed foruse in a vaccine. One of the genes encodes phospholipase B, which can be expressed with high purity and yield in recombinant Escherichiacoli as a secreted, soluble, non-tagged, mature construct (less signal peptide with predicted mass 63 kDa). The recombinant phospholi-pase B exhibited anomalous electrophoretic mobility that was dependent on the temperature of the denaturing process, with bandsobserved at either 52 or 63 kDa. Analysis by in-gel digestion and liquid chromatography–mass spectrometry revealed these two distinctforms most likely had identical sequences. Phospholipase B is a compact, globular protein with a predicted structure typical of a con-ventional autotransporter. It is suggested that high temperature is required to unfold the protein (to denature the b-barrel-rich trans-porter domain) and to ensure accessibility of the reducing agent. Interestingly, the two forms of the enzyme, differing in size andisoelectric points, were also detected in cell-free supernatants of M. bovis cultures, indicating that native phospholipase B may existin two differentially folded states possibly also differing in oxidation status of cysteine residues.Crown copyright 2007 Published by Elsevier Inc. All rights reserved. |
| Databáze: | OpenAIRE |
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