Partial characterization of the 30 kD Ig-binding protein from Pseudomonas maltophilia.
We have previously demonstrated that Pseudomonas maltophilia (ATCC 13637) possess a 30 kDa cell wall protein which binds various subclasses of IgG's and IgA by their Fc region. The protein was solubilized by papain and purified by affinity chromatography on cyanogen bromide activated sepharose beads conjugated with human IgG. The eluent was electrophoresed on a 12% polyacrylamide gel under denaturing conditions, and the immunoactive bands identified by Western blot analysis, a second gel was stained with Coomassie blue. The affinity purified eluent was electrophoresed on a one-dimensional 15% polyacrylamide gel and stained with Coomassie blue. The protein band of interest was cut. The protein band was then digested in situ with Staphylococcus aureus V-8 protease. The peptide bands were separated by electrophoresis on a second one dimensional 15% polyacrylamide gel and then electroblotted into a polyvinylidine difluoride membrane. The bands were visualized by staining with Coomassie blue, cut out, and sequenced using an automated gas phase sequencer. Minimal amino acid composition was determined in a similar fashion. We have thus obtained partial N-terminal amino acid sequence data from the above method.
Grover
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Department of Medicine and Physiology, University of Utah School of Medicine, Salt Lake City 84132.
Odell
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WD
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D008222:Lymphokines / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D029584:Prostatic Secretory Proteins; D011549:Pseudomonas / Q000378:metabolism*; D012697:Serine Endopeptidases
D008222:Lymphokines; D010446:Peptide Fragments; D029584:Prostatic Secretory Proteins; C043076:beta-microseminoprotein; C013059:immunoglobulin-binding factors; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase