title: Application of simple fed-batch technique to high-level secretory production of insulin precursor using Pichia pastoris with subsequent purification and conversion to human insulin.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  A synthetic insulin precursor (IP)-encoding gene, codon-optimized for expression in P. pastoris, was cloned in frame with the Saccharomyces cerevisiae alpha-factor secretory signal and integrated into the genome of P. pastoris 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-1, which were kept constant throughout the remaining production phase. This robust feeding strategy led to the secretion of approximately 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 approximately 1.5 g of 99% pure recombinant human insulin per liter of culture broth.
  
  
  CONCLUSIONS
  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 Pichia based expression systems, thus significantly increasing the efficiency of insulin manufacture.
authors.affiliation: Helmholtz Centre for Infection Research, Braunschweig, Germany.
authors.forename: Chandrasekhar
authors.initials: C
authors.lastname: Gurramkonda
authors.forename: Sulena
authors.initials: S
authors.lastname: Polez
authors.forename: Natasa
authors.initials: N
authors.lastname: Skoko
authors.forename: Ahmad
authors.initials: A
authors.lastname: Adnan
authors.forename: Thomas
authors.initials: T
authors.lastname: Gäbel
authors.forename: Dipti
authors.initials: D
authors.lastname: Chugh
authors.forename: Sathyamangalam
authors.initials: S
authors.lastname: Swaminathan
authors.forename: Navin
authors.initials: N
authors.lastname: Khanna
authors.forename: Sergio
authors.initials: S
authors.lastname: Tisminetzky
authors.forename: Ursula
authors.initials: U
authors.lastname: Rinas
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D003470:Culture Media; D005990:Glycerol / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D000078790:Insulin Secretion; D000432:Methanol / Q000378:metabolism; D010843:Pichia / Q000235:genetics*; D013678:Technology, Pharmaceutical / Q000379:methods*
chemical_list: D003470:Culture Media; D007328:Insulin; D005990:Glycerol; D000432:Methanol
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