Protein production method

A method of producing a heterologous polypeptide, the method comprising the steps of:

• a) growing Gram-negative bacteria comprising a nucleotide sequence encoding a heterologous polypeptide operably linked to an inducible promoter under fed-batch fermentation conditions in a synthetic medium;
• b) inducing expression of the heterologous polypeptide;
• c) harvesting the bacteria in the synthetic medium by decanting;
• d) homogenizing the bacteria contained in the synthetic medium;
• e) obtaining from the synthetic medium comprising the homogenized bacteria of step d) a soluble fraction comprising the heterologous polypeptide and an insoluble fraction comprising the heterologous polypeptide;
• f) resuspending the insoluble fraction;
• g) centrifuging the resuspended insoluble fraction of step g) to obtain a supernatant comprising the heterologous polypeptide;
• h) denaturing the polypeptide in the supernatant obtained from step g);
• i) refolding the denatured polypeptide of step h);
• and
• j) subjecting the refolded polypeptide to anion exchange chromatography to obtain the heterologous polypeptide.