2. Academic Validation
  3. High level expression and purification of active recombinant human interleukin-15 in Pichia pastoris

High level expression and purification of active recombinant human interleukin-15 in Pichia pastoris

  • J Immunol Methods. 2016 Jan;428:50-7. doi: 10.1016/j.jim.2015.12.002.
Wei Sun 1,  Yunxin Lai 2,  Hongbo Li 3,  Tao Nie 2,  Ye Kuang 4,  Xiaofeng Tang 2,  Kuai Li 2,  P Rod Dunbar 5,  Aimin Xu 6,  Peng Li 7,  Donghai Wu 8
Affiliations

Affiliations

  • 1 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.
  • 2 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.
  • 3 Key Laboratory of Research and Utilization of Ethnomedicinal Plant Resources of Hunan Province, Department of Life Sciences, Huaihua College, Huaihua, China.
  • 4 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.
  • 5 School of Biological Sciences & Maurice Wilkins Centre, University of Auckland, Auckland, New Zealand.
  • 6 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.
  • 7 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: [email protected].
  • 8 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: [email protected].
PMID: 26678161 DOI: 10.1016/j.jim.2015.12.002
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.

Keywords

Expression and purification; Interleukin-15; N-linked glycosylation; Pichia pastoris.

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