Interleukin-3 (IL-3) is a hematopoietic growth factor involved in the survival, proliferation and differentiation of multipotent hematopoietic cells. A DNA fragment containing the mature human IL-3 sequence was cloned into pPICZαA, 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 rhIL-3 production were identified, secreting as much as 26mg/L rhIL-3 after 4days of induction by methanol in flask. The rhIL-3 was purified by Ni(+)-NTA affinity chromatography, followed by DEAE anion exchange, yielding over 95% highly purified rhIL-3 preparation at about 21mg/L. Mass spectrometry and MALDI-TOF-TOF analysis of the purified rIL-3 showed molecular weights of 18995.694Da and 22317.469Da, due to different degrees of N-linked glycosylation. The biological activity of the rhIL-3 proteins was confirmed by its ability to support ba/f3 cells proliferation and activate the ERK signaling pathways. The results demonstrate that the experimental procedure we have developed can produce a large amount of active recombinant human IL-3 from P. pastoris.
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