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Transferrin receptor protein of moraxella
| Details |
Inventors: Yang, Yan-Ping; Myers, Lisa E.; Harkness, Robin E.; Klein, Michel H.;
Assignee: Connaught Laboratories Limited (Toronto, CA)
Primary Examiner: Minnifield; Nita
Assistant Examiner:
Attorney, Agent or Firm: Sim & McBurney
An isolated and purified non-denatured transferrin receptor protein of a Moraxella strain, particularly M. catarrhalis, has an apparent molecular mass of about 80 to about 90 kDa, as determined by SDS-PAGE. The transferrin receptor protein or a fragment analog thereof is useful in diagnostic applications and immunogenic compositions, particularly for in vivo administration to a host to confer protection against disease caused by a strain of Moraxella. The transferrin receptor protein is isolated from strains of Moraxella catarrhalis by a procedure including extraction of agent soluble proteins of a cell mass produced by cultivating the strain under iron-starved conditions. The transferrin receptor protein is selectively solubilized from the extracted cell mass and purified. |
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DETAILED DESCRIPTION What we claim is: 1. A method of producing an isolated and purified non-denatured transferrin receptor protein of a Moraxella catarrhalis strain having an apparent molecular mass of about 80 to about 90 kDa, comprising the steps of: (a) providing a cell mass of the Moraxella catarrhalis strain grown under iron-starved conditions to stimulate transferrin receptor protein expression; (b) selectively extracting aqueous soluble proteins from said cell mass by contacting the cell mass with a first buffered aqueous solution sonicating the cell mass to disrupt the same, and centrifuging to provide a first supernatant and a first pellet; (c) separating said first supernatant from said first pellet; (d) selectively solubilizing at least a transferrin receptor protein having a molecular mass of about 80 to 90 kDa from said first pellet to provide a second supernatant and a second pellet by contacting the first pellet at least once with a second buffered aqueous solution comprising a detergent and a stabilization agent, sonication of said first pellet to disrupt the same and centrifuging to provide the second supernatant and the second pellet; (e) separating said second supernatant from said second pellet; and (f) purifying transferrin receptor protein having a molecular mass of about 80 to 90 kDa in said second supernatant free from other Moraxella catarrhalis proteins solubilized from said first pellet in said selective solubilization step by multiple chromatographic operations, including: i) a first chromatographic operation on a first chromatography column through which said transferrin receptor protein selectively flows and on which contaminating proteins bind, ii) a second chromatographic operation on a second chromatography column comprising a cation-exchange matrix through which said transferrin receptor protein selectively flows and on which contaminating proteins bind; iii) a third chromatographic operation on a third chromatography column on which said transferrin receptor protein is selectively bound in preference to the OMP B2 protein of the Moraxella catarrhalis strain; and iv) eluting said transferrin receptor protein from said third chromatography column
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