Efficient expression and isolation of recombinant human interleukin-11 (rhIL-11) in Pichia pastoris.

Autor: Yu KM; Nansha Biologics (Hong Kong) Limited, Unit 608-613, IC Development Centre, No.6 Science Park West Avenue, Hong Kong Science Park, Shatin, Hong Kong Special Administrative Region, China; Department of Applied Biology & Chemical Technology, The Hong Kong Polytechnic University, 11 Yuk Choi Road, Hung Hom, Kowloon, Hong Kong. Electronic address: kyu@nansha-biologics.com., Yiu-Nam Lau J; Nansha Biologics (Hong Kong) Limited, Unit 608-613, IC Development Centre, No.6 Science Park West Avenue, Hong Kong Science Park, Shatin, Hong Kong Special Administrative Region, China., Fok M; Nansha Biologics (Hong Kong) Limited, Unit 608-613, IC Development Centre, No.6 Science Park West Avenue, Hong Kong Science Park, Shatin, Hong Kong Special Administrative Region, China., Yeung YK; Nansha Biologics (Hong Kong) Limited, Unit 608-613, IC Development Centre, No.6 Science Park West Avenue, Hong Kong Science Park, Shatin, Hong Kong Special Administrative Region, China., Fok SP; Nansha Biologics (Hong Kong) Limited, Unit 608-613, IC Development Centre, No.6 Science Park West Avenue, Hong Kong Science Park, Shatin, Hong Kong Special Administrative Region, China., Shek F; Nansha Biologics (Hong Kong) Limited, Unit 608-613, IC Development Centre, No.6 Science Park West Avenue, Hong Kong Science Park, Shatin, Hong Kong Special Administrative Region, China., Wong WT; Department of Applied Biology & Chemical Technology, The Hong Kong Polytechnic University, 11 Yuk Choi Road, Hung Hom, Kowloon, Hong Kong., Choo QL; Nansha Biologics (Hong Kong) Limited, Unit 608-613, IC Development Centre, No.6 Science Park West Avenue, Hong Kong Science Park, Shatin, Hong Kong Special Administrative Region, China.
Jazyk: angličtina
Zdroj: Protein expression and purification [Protein Expr Purif] 2018 Jun; Vol. 146, pp. 69-77. Date of Electronic Publication: 2018 Feb 03.
DOI: 10.1016/j.pep.2018.01.012
Abstrakt: Current source of recombinant human interleukin-11 (rhIL-11) is isolated from a fusion protein expressed by E. coli that requires additional enterokinase to remove linked protein, resulting in product heterogeneity of N-terminal sequence. Due to lack of glycosylation, rhIL-11 is suitable to be expressed by yeast cells. However, the only available yeast-derived rhIL-11 presents an obstacle in low production yield, as well as an unamiable process, such as the use of reverse-phase chromatography employing plenty of toxic organic solvents. Our findings showed that the low yield was due to self-aggregation of rhIL-11. A novel process recovering bioactive rhIL-11 from the yeast secretory medium therefore has been developed and demonstrated, involving fermentation from Pichia pastoris, followed by a two-phase extraction to precipitate rhIL-11. After renaturing, the protein of interest was purified by a two-column step, comprising a cation-exchanger, and a hydrophobic interaction chromatography in tandem at high sample loads that was facile and cost-effective in future scale-up. Identity and quality assessments confirmed the expected amino acid sequence without N-terminal heterogeneity, as well as high quality in potency and purity. Such a process provides an alternative and adequate supply of the starting material for the PEGylated rhIL-11.
(Copyright © 2018 Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
Externí odkaz: