title: Efficient expression and secretion of recombinant human growth hormone in the methylotrophic yeast Pichia pastoris: potential applications for other proteins.
abstract: A simple high yielding process for the production of rhGH (recombinant human growth hormone) in the Pichia pastoris system is described. The approach adopted the addition of surfactants during fermentation along with methanol induction. A Pichia integrant harbouring multiple-copy, non-codon-optimized hGH showed poor expression in complex and defined media. Inclusion of the surfactants Tween 20 or Tween 80 during induction enhanced the expression levels significantly in shake flask studies. The combination of 2 litres of basal salt medium and Tween 20 in a bioreactor culminated in 3 x 10(4)-fold elevated expression of the protein (approximately 500 mg/l) as estimated by ELISA. SDS/PAGE and Western-blot analyses revealed that the Pichia-derived rhGH migrated as a single band with a molecular mass of approximately 22 kDa and had the same immunoreactivity as native commercial rhGH. Analysis of Pichia-derived purified rhGH and commercial rhGH on an Agilent 2100 Bioanalyzer revealed overlapping peaks displaying authentic N-terminal processing of Pichia-derived rhGH, which was further confirmed by N-terminal sequencing. In addition, matrix-assisted laser-desorption ionization-time of flight analysis of the protein confirmed its authenticity. These results indicate that the P. pastoris expression system can be effective in the production of rhGH at commercially relevant levels.
authors.affiliation: Clone Development Team, Lupin Limited, Biotechnology R&D, Pune, India.
authors.forename: Anjali
authors.initials: A
authors.lastname: Apte-Deshpande
authors.forename: Sachin
authors.initials: S
authors.lastname: Rewanwar
authors.forename: Prakash
authors.initials: P
authors.lastname: Kotwal
authors.forename: Veena A
authors.initials: VA
authors.lastname: Raiker
authors.forename: Sriram
authors.initials: S
authors.lastname: Padmanabhan
mesh_terms: D019149:Bioreactors; D003001:Cloning, Molecular; D005285:Fermentation; D018628:Gene Dosage; D015870:Gene Expression; D005822:Genetic Vectors; D019382:Human Growth Hormone / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics*; D006801:Humans; D010843:Pichia / Q000235:genetics* / Q000254:growth & development; D011136:Polysorbates / Q000378:metabolism
chemical_list: D011136:Polysorbates; D019382:Human Growth Hormone