| Summary: | <p>Abstract</p> <p>Background</p> <p>The prevalence of diabetes is predicted to rise significantly in the coming decades. A recent analysis projects that by the year 2030 there will be ~366 million diabetics around the world, leading to an increased demand for inexpensive insulin to make this life-saving drug also affordable for resource poor countries.</p> <p>Results</p> <p>A synthetic insulin precursor (IP)-encoding gene, codon-optimized for expression in <it>P. pastoris</it>, was cloned in frame with the <it>Saccharomyces cerevisiae </it>α-factor secretory signal and integrated into the genome of <it>P. pastoris </it>strain X-33. The strain was grown to high-cell density in a batch procedure using a defined medium with low salt and high glycerol concentrations. Following batch growth, production of IP was carried out at methanol concentrations of 2 g L<sup>-1</sup>, which were kept constant throughout the remaining production phase. This robust feeding strategy led to the secretion of ~3 gram IP per liter of culture broth (corresponding to almost 4 gram IP per liter of cell-free culture supernatant). Using immobilized metal ion affinity chromatography (IMAC) as a novel approach for IP purification, 95% of the secreted product was recovered with a purity of 96% from the clarified culture supernatant. Finally, the purified IP was trypsin digested, transpeptidated, deprotected and further purified leading to ~1.5 g of 99% pure recombinant human insulin per liter of culture broth.</p> <p>Conclusions</p> <p>A simple two-phase cultivation process composed of a glycerol batch and a constant methanol fed-batch phase recently developed for the intracellular production of the Hepatitis B surface antigen was adapted to secretory IP production. Compared to the highest previously reported value, this approach resulted in an ~2 fold enhancement of IP production using <it>Pichia </it>based expression systems, thus significantly increasing the efficiency of insulin manufacture.</p>
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