title: High level expression of active recombinant human interleukin-3 in Pichia pastoris.
abstract: 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.
authors: The Key Laboratory of Regenerative Biology, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China.
authors: Hongbo
authors: H
authors: Li
authors: Na
authors: N
authors: Li
authors: Xuefei
authors: X
authors: Gao
authors: Xiangping
authors: X
authors: Kong
authors: Shiwu
authors: S
authors: Li
authors: Aimin
authors: A
authors: Xu
authors: Shouguang
authors: S
authors: Jin
authors: Donghai
authors: D
authors: Wu
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D005285:Fermentation; D006031:Glycosylation; D006801:Humans; D007377:Interleukin-3 / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D020935:MAP Kinase Signaling System; D013058:Mass Spectrometry; D051379:Mice; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014170:Transformation, Genetic
chemical_list: C508596:IL3 protein, human; D007377:Interleukin-3; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins