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. Epub 2015 Dec 8.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line
  • Cell Proliferation
  • Humans
  • Interleukin-15 / biosynthesis
  • Interleukin-15 / genetics
  • Interleukin-15 / immunology*
  • Interleukin-15 / isolation & purification*
  • Killer Cells, Natural / cytology
  • Killer Cells, Natural / immunology
  • Mice
  • Mice, Inbred NOD
  • Mice, Knockout
  • Mice, SCID
  • Pichia / genetics*
  • Recombinant Proteins / biosynthesis*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification*

Substances

  • IL15 protein, human
  • Interleukin-15
  • Recombinant Proteins