Method for producing purified recombinant Haemophilus influenzae transferrin binding proteins

A method of producing an isolated and purified Tbp1 or Tbp2 protein of a strain of Haemophilus influenzae, comprising the steps of:

• (a) providing a recombinant host expressing, in inclusion bodies, a Tbp1 or Tbp2 protein, but not both, of a strain of Haemophilus influenzae;
• (b) growing said host to provide a cell mass wherein said Tbp1 or Tbp2 protein is present in inclusion bodies;
• (c) disrupting the cell mass to provide a cell lysate;
• (d) fractionating the cell lysate to provide a first supernatant and a first pellet, the first supernatant comprising substantially a large proportion of soluble host proteins;
• (e) separating said first supernatant from said first pellet;
• (f) selectively extracting the first pellet to remove substantially all soluble host proteins and host membrane proteins therefrom to provide a second supernatant and an extracted pellet containing the inclusion bodies;
• (g) separating said second supernatant from said extracted pellet;
• (h) solubilizing the extracted pellet to provide a solubilized extract;
• and
• (i) fractionating the solubilized extract to provide a Tbp1 or Tbp2 protein containing fraction.