Patent ID: 6190668
Filing Date: 2001-02-20
Classification: A61K,A61P,C07K

Abstract:
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; andiv) eluting said transferrin receptor protein from said third chromatography column.