title: High level expression and purification of active recombinant human interleukin-15 in Pichia pastoris.
abstract: Interleukin-15 (IL-15) is a pleiotropic cytokine and a member of the four α-helix bundle family of cytokines which include IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21. IL-15 exhibits a broad biological activity and induces the differentiation and proliferation of T, B and natural killer (NK) cells. In this study, a DNA fragment containing the mature human IL-15 sequence was cloned into pPICZaA vector, generating a fusion protein with the alpha factor signal sequence in the N-terminus and 6×His as well as c-Myc tags in the C-terminus. The resulting plasmid was integrated into the genome of Pichia pastoris strain X-33. Recombinant yeast transformants with high-level recombinant human IL-15 (rhIL-15) production were identified, which secrete as much as 75 mg/L rhIL-15 after 3 days of induction by methanol. The rhIL-15 was purified by Ni(+)-NTA affinity chromatography, followed by DEAE anion exchange, yielding over 95% highly purified rhIL-15. Mass spectrometry and MALDI-TOF-TOF analysis showed the purified rhIL-15 had larger molecular weights than expected, due to different degrees of N-linked glycosylation. The biological activity of the rhIL-15 proteins was measured by its ability to enhance cellular proliferation of CTLL-2 and NK cells. The results demonstrate that the experimental procedure we have reported here can produce a large amount of active recombinant human IL-15 from P. pastoris.
authors.affiliation: The Key Laboratory of Regenerative Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences and Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou, 510530, China; School of Life Sciences, Anhui University, Hefei 230601, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Sun
authors.affiliation: The Key Laboratory of Regenerative Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences and Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou, 510530, China.
authors.forename: Yunxin
authors.initials: Y
authors.lastname: Lai
authors.affiliation: Key Laboratory of Research and Utilization of Ethnomedicinal Plant Resources of Hunan Province, Department of Life Sciences, Huaihua College, Huaihua, China.
authors.forename: Hongbo
authors.initials: H
authors.lastname: Li
authors.affiliation: The Key Laboratory of Regenerative Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences and Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou, 510530, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Nie
authors.affiliation: The Key Laboratory of Regenerative Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences and Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou, 510530, China; Department of Biomedical Engineering, School of Pharmaceutical Sciences, Jilin University, Changchun, China.
authors.forename: Ye
authors.initials: Y
authors.lastname: Kuang
authors.affiliation: The Key Laboratory of Regenerative Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences and Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou, 510530, China.
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Tang
authors.affiliation: The Key Laboratory of Regenerative Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences and Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou, 510530, China.
authors.forename: Kuai
authors.initials: K
authors.lastname: Li
authors.affiliation: School of Biological Sciences & Maurice Wilkins Centre, University of Auckland, Auckland, New Zealand.
authors.forename: P Rod
authors.initials: PR
authors.lastname: Dunbar
authors.affiliation: The Key Laboratory of Regenerative Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences and Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou, 510530, China; Department of Pharmacology and Medicine, The University of Hong Kong, Hong Kong, China.
authors.forename: Aimin
authors.initials: A
authors.lastname: Xu
authors.affiliation: The Key Laboratory of Regenerative Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences and Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou, 510530, China. Electronic address: li_peng@gibh.ac.cn.
authors.forename: Peng
authors.initials: P
authors.lastname: Li
authors.affiliation: The Key Laboratory of Regenerative Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences and Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou, 510530, China. Electronic address: wu_donghai@gibh.ac.cn.
authors.forename: Donghai
authors.initials: D
authors.lastname: Wu
mesh_terms: D000818:Animals; D002460:Cell Line; D049109:Cell Proliferation; D006801:Humans; D019409:Interleukin-15 / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification*; D007694:Killer Cells, Natural / Q000166:cytology / Q000276:immunology; D051379:Mice; D016688:Mice, Inbred NOD; D018345:Mice, Knockout; D016513:Mice, SCID; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*
chemical_list: C569274:IL15 protein, human; D019409:Interleukin-15; D011994:Recombinant Proteins
keywords: Expression and purification; Interleukin-15; N-linked glycosylation; Pichia pastoris