title: Expression and Control of Codon-Optimized Granulocyte Colony-Stimulating Factor in Pichia pastoris.
abstract: Granulocyte colony-stimulating factor (GCSF) has therapeutic applications due to its proven efficacy in different forms of neutropenia and chemotherapy-induced leucopenia. The original 564-bp nucleotide sequence from NCBI was codon optimized and assembled by overlapping PCR method comprising of 16 oligos of 50-nt length with 15 base overhang. The synthetic gene (CO-GCSF) was cloned under glucose utilizing glyceraldehyde 3-phosphate dehydrogenase (GAP) and methanol-utilizing alcohol oxidase (AOX1) promoters and expressed in Pichia pastoris SMD1168 strain. Constitutive expression under GAP resulted in cellular toxicity while AOX1 promoter controlled expression was stable. Variation in the levels of expression was observed among the transformant colonies with transformant #2 secreting up to ∼4 mg/L of GCSF. The molecular mass of the expressed GCSF in P. pastoris was ∼19.0 kDa. Quatitation of the expressed protein was carried out by a highly reproducible gel densitometric method. Effect of several operational and nutritional conditions was studied on GCSF production and the results suggest a general approach for increasing the yield of GCSF several folds (2- to 5-fold) over the standard conditions employed currently. Cultivation of the single-copy integrant in the chemically defined medium in a 5-L fermenter resulted in a volumetric productivity of ∼0.7 mg/L/h at the end of the induction phase, which was about 4-fold higher than attained in the shake flask.
authors.affiliation: Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi, 110016, India.
authors.forename: Nitu
authors.initials: N
authors.lastname: Maity
authors.affiliation: Lilly Hall of Biological Sciences, Purdue University - West Lafayette, Indiana, USA.
authors.forename: Ankita
authors.initials: A
authors.lastname: Thawani
authors.affiliation: Biochemical Engineering Department, University College London, London, UK.
authors.forename: Anshul
authors.initials: A
authors.lastname: Sharma
authors.affiliation: Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi, 110016, India.
authors.forename: Ashwani
authors.initials: A
authors.lastname: Gautam
authors.affiliation: Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi, 110016, India. saroj98@hotmail.com.
authors.forename: Saroj
authors.initials: S
authors.lastname: Mishra
authors.affiliation: Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi, 110016, India.
authors.forename: Vikram
authors.initials: V
authors.lastname: Sahai
mesh_terms: D019149:Bioreactors; D003001:Cloning, Molecular; D003062:Codon; D016179:Granulocyte Colony-Stimulating Factor / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D016366:Open Reading Frames; D010843:Pichia / Q000235:genetics*
chemical_list: D003062:Codon; D016179:Granulocyte Colony-Stimulating Factor
keywords: AOX1 promoter; Amino acid supplementation; Codon-optimized GCSF; Low pH stability; Pichia pastoris