Premium
RSM optimization of HSA/IL1Ra in Pichia pastoris overexpression strain and study of its in vivo activity in reducing hyperglycemia of GK rats
Author(s) -
Liu Wenhui,
Zhao Wenbin,
Lai Jun,
Shen Qi,
Xu Yingchun,
Pan Liqiang,
Chen Shuqing
Publication year - 2017
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1002/bab.1532
Subject(s) - pichia pastoris , interleukin 1 receptor antagonist , industrial fermentation , in vivo , antagonist , fermentation , biology , human serum albumin , receptor antagonist , biochemistry , recombinant dna , receptor , gene , microbiology and biotechnology
Human serum albumin (HSA) and interleukin‐1 receptor antagonist (IL1Ra) fusion protein is a potential long‐acting drug in the treatment of type 2 diabetes. Previously, the expression level of HSA/IL1Ra in Pichia pastoris was successfully improved by increasing the gene copy number and coexpression with chaperone (protein disulfide isomerase) in our laboratory. However, the overexpression strain resulted in low production of high‐ cell‐density fermentation. In this study, the culture medium was optimized in both flask and fermenter, and the optimum culture medium notably increased the productivity and stability of HSA/IL1Ra. To further improve the expression, response surface methodology was used to further optimize the culture condition through modeling three selected parameters (induction pH, induction temperature [ T ], and maximum methanol feed rate [ V m ]). The maximum yield of HSA/IL1Ra reached 1.1 g/L (10‐fold higher than original fermentation condition) under the optimized culture condition (pH 7.0, T = 29 ℃ and V m = 4.82 mL/L/H) in a 5‐L fermenter. In addition, the degradation position of HSA/IL1Ra during fermentation was determined to be K571, serving as a potential target for genetic modification strategies to reduce the degradation. Finally, the in vivo activity study showed that HSA/IL1Ra maintained the therapeutic effect of IL1Ra in type 2 diabetes model rats meanwhile reducing the frequency of administration.