Expression and Control of Codon-Optimized Granulocyte Colony-Stimulating Factor in Pichia pastoris.
Appl Biochem Biotechnol
; 178(1): 159-72, 2016 Jan.
Article
em En
| MEDLINE
| ID: mdl-26410223
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 â¼4 mg/L of GCSF. The molecular mass of the expressed GCSF in P. pastoris was â¼19.0 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 â¼0.7 mg/L/h at the end of the induction phase, which was about 4-fold higher than attained in the shake flask.
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Base de dados:
MEDLINE
Assunto principal:
Pichia
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Códon
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Fator Estimulador de Colônias de Granulócitos
Idioma:
En
Ano de publicação:
2016
Tipo de documento:
Article