title: RSM optimization of HSA/IL1Ra in Pichia pastoris overexpression strain and study of its in vivo activity in reducing hyperglycemia of GK rats.
abstract: 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 [Vm]). 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 Vm= 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.
authors: Department of Drug Metabolism and Drug Analysis, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, People's Republic of China.
authors: Wenhui
authors: W
authors: Liu
authors: Department of Drug Metabolism and Drug Analysis, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, People's Republic of China.
authors: Wenbin
authors: W
authors: Zhao
authors: Department of Drug Metabolism and Drug Analysis, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, People's Republic of China.
authors: Jun
authors: J
authors: Lai
authors: Department of Nanomedicine, Houston Methodist Research Institute, Houston, TX, USA.
authors: Qi
authors: Q
authors: Shen
authors: Department of Drug Metabolism and Drug Analysis, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, People's Republic of China.
authors: Yingchun
authors: Y
authors: Xu
authors: Department of Drug Metabolism and Drug Analysis, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, People's Republic of China.
authors: Liqiang
authors: L
authors: Pan
authors: Department of Drug Metabolism and Drug Analysis, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, People's Republic of China.
authors: Shuqing
authors: S
authors: Chen
mesh_terms: D000818:Animals; D001786:Blood Glucose / Q000187:drug effects; D003924:Diabetes Mellitus, Type 2; D006801:Humans; D006943:Hyperglycemia; D007004:Hypoglycemic Agents / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology; D053590:Interleukin 1 Receptor Antagonist Protein / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology; D012107:Research Design; D012709:Serum Albumin / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology
chemical_list: D001786:Blood Glucose; D007004:Hypoglycemic Agents; C507763:IL1RN protein, human; D053590:Interleukin 1 Receptor Antagonist Protein; D011993:Recombinant Fusion Proteins; D012709:Serum Albumin
keywords: HSA fusion protein; interleukin-1 receptor antagonist; response surface methodology; type 2 diabetes