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

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• 作者：Wei Sun^a ; ^b ; Yunxin Lai^a ; Hongbo Li^c ; Tao Nie^a ; Ye Kuang^a ; ^d ; Xiaofeng Tang^a ; Kuai Li^a ; P. Rod Dunbar^e ; Aimin Xu^a ; ^f ; Peng Li^a ; ^{li_peng@gibh.ac.cn" class="auth_mail" title="E-mail the corresponding author} ; Donghai Wu^a ; ^{wu_donghai@gibh.ac.cn" class="auth_mail" title="E-mail the corresponding author}
• 关键词：Interleukin-15 ; Pichia pastoris ; Expression and purification ; N-linked glycosylation
• 刊名：Journal of Immunological Methods
• 出版年：2016
• 出版时间：January 2016
• 年：2016
• 卷：428
• 期：Complete
• 页码：50-57
• 全文大小：1079 K

文摘

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.