High Level Expression And Purification Of Active Recombinant Human Interleukin-15 In Pichia Pastoris

Interleukin-15 (IL-15) is a pleiotropic cytokine and a member of the four α-helix bundle family of protein factors which also include IL-2, IL-4, IL-7, IL-9, and IL-21. Human IL-15 gene maps to chromosome 4q31 and consists of eight protein-coding exons and five introns. IL-15 is a 14-15-kDa cytokine stringently controlled and expressed primarily within monocytes, macrophages, and dendritic cells.IL-15 exhibits a broad biological activity and induces the differentiation and proliferation of T, B and natural killer (NK) cells. IL-15 can play pleiotropic roles in immune responses in infection、 tumor、allergy、and autoimmune diseases. 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 at the N-terminus and 6 X His as well as c-Myc tags at the C-terminus. The resulting plasmid was integrated into the genome of Pichia pastoris strain X-33. 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 4 days of induction by methanol. The rhIL-15 was purified by Ni+-NTA affinity chromatography, followed by DEAE anion exchange column, 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 was measured by its ability to enhance cellular proliferation of CTLL-2 and NK cells. The results demonstrated that the experimental procedure we have reported here can produce a large amount of active recombinant human IL-15 from Pichia pastoris.