title: The effect of gene copy number and co-expression of chaperone on production of albumin fusion proteins in Pichia pastoris.
abstract: Interleukin-1 receptor antagonist (IL1ra) is known to treat a number of diseases such as rheumatoid arthritis and type 2 diabetes. However, the biological half-life of IL1ra is very short due to its rapid renal clearance. Our present study aimed to increase the biological half-life of IL1ra through fusion with human serum albumin (HSA), and then augmented expression of the IL1ra and HSA fusion protein (IH) in Pichia pastoris strain by increasing IH gene copy number or was co-expressed with chaperone. By comparing clones containing varying copy numbers of IH fusion gene, it was observed that higher levels of secretory IH fusion protein was produced in strain with higher IH gene copy number. In addition, IH protein yield was further improved after being co-expressed with protein disulfide isomerase (PDI). Conversely, it was significantly decreased (i.e., secretory IH in the culture medium) by co-expression of immunoglobulin binding protein. We have also discussed whether the multi-copy strain and co-expressed of PDI could enhance the levels of other secretory albumin fusion protein (e.g., HSA and human growth hormone fusion protein). Interestingly, the level of this fusion protein was apparently also increased by these approaches. In conclusion, our results have demonstrated that increasing copy number and co-expression of PDI may raise yield of albumin fusion protein in P. pastoris, which might probably contribute to the industry for the development of proteinous drugs.
authors.affiliation: Department of Pharmacology, Toxicology and Biochemical Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University Hangzhou, Hangzhou, 310058, China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Shen
authors.forename: Min
authors.initials: M
authors.lastname: Wu
authors.forename: Ming
authors.initials: M
authors.lastname: Wu
authors.forename: Hai-Bin
authors.initials: HB
authors.lastname: Wang
authors.forename: Hua
authors.initials: H
authors.lastname: Naranmandura
authors.forename: Shu-Qing
authors.initials: SQ
authors.lastname: Chen
mesh_terms: D001709:Biotechnology / Q000379:methods; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D018628:Gene Dosage; D053590:Interleukin 1 Receptor Antagonist Protein / Q000235:genetics / Q000378:metabolism*; D060847:Metabolic Engineering; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D012709:Serum Albumin / Q000235:genetics / Q000378:metabolism*; D000075462:Serum Albumin, Human
chemical_list: C422455:ALB protein, human; D004271:DNA, Fungal; C507763:IL1RN protein, human; D053590:Interleukin 1 Receptor Antagonist Protein; D018832:Molecular Chaperones; D011993:Recombinant Fusion Proteins; D012709:Serum Albumin; D000075462:Serum Albumin, Human