Abstract
DNA-sequences coding for two DesPro(2) aprotinin variants were expressed in the methylotrophic yeast Hansenula polymorpha from a strong inducible promoter element derived from the MOX gene, a key gene of methanol metabolism. For secretion the coding sequences were fused to the KEX2 recognition site of the S. cerevisiae-derived MFα1 preproleader sequence. Correct processing of the precursor molecules and efficient secretion of the mature proteins was observed. A pH/pO 2-controlled C-source feeding mode was applied for fermentations on a 10 litre scale. In cultures of a transformant strain harbouring 20 copies of the aprotinin expression cassette a yield of 350 mg/litre could be obtained. The secreted recombinant products were purified in a simple two-step isolation procedure employing an expanded bed adsorption as an initial purification step.
Published Version
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