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- Publisher Website: 10.1016/j.pep.2011.08.027
- Scopus: eid_2-s2.0-80052845786
- PMID: 21907288
- WOS: WOS:000296225800004
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Article: High level expression of active recombinant human interleukin-3 in Pichia pastoris
Title | High level expression of active recombinant human interleukin-3 in Pichia pastoris |
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Authors | |
Keywords | Expression and purification His×6-tag Interleukin-3 N-linked glycosylation Pichia pastoris |
Issue Date | 2011 |
Publisher | Academic Press. The Journal's web site is located at http://www.elsevier.com/locate/yprep |
Citation | Protein Expression And Purification, 2011, v. 80 n. 2, p. 185-193 How to Cite? |
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 26 mg/L rhIL-3 after 4 days 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 21 mg/L. Mass spectrometry and MALDI-TOF-TOF analysis of the purified rIL-3 showed molecular weights of 18995.694 Da and 22317.469 Da, 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. © 2011 Elsevier Inc. All rights reserved. |
Persistent Identifier | http://hdl.handle.net/10722/163399 |
ISSN | 2023 Impact Factor: 1.4 2023 SCImago Journal Rankings: 0.383 |
ISI Accession Number ID | |
References |
DC Field | Value | Language |
---|---|---|
dc.contributor.author | Li, H | en_US |
dc.contributor.author | Li, N | en_US |
dc.contributor.author | Gao, X | en_US |
dc.contributor.author | Kong, X | en_US |
dc.contributor.author | Li, S | en_US |
dc.contributor.author | Xu, A | en_US |
dc.contributor.author | Jin, S | en_US |
dc.contributor.author | Wu, D | en_US |
dc.date.accessioned | 2012-09-05T05:30:54Z | - |
dc.date.available | 2012-09-05T05:30:54Z | - |
dc.date.issued | 2011 | en_US |
dc.identifier.citation | Protein Expression And Purification, 2011, v. 80 n. 2, p. 185-193 | en_US |
dc.identifier.issn | 1046-5928 | en_US |
dc.identifier.uri | http://hdl.handle.net/10722/163399 | - |
dc.description.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 26 mg/L rhIL-3 after 4 days 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 21 mg/L. Mass spectrometry and MALDI-TOF-TOF analysis of the purified rIL-3 showed molecular weights of 18995.694 Da and 22317.469 Da, 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. © 2011 Elsevier Inc. All rights reserved. | en_US |
dc.language | eng | en_US |
dc.publisher | Academic Press. The Journal's web site is located at http://www.elsevier.com/locate/yprep | en_US |
dc.relation.ispartof | Protein Expression and Purification | en_US |
dc.subject | Expression and purification | - |
dc.subject | His×6-tag | - |
dc.subject | Interleukin-3 | - |
dc.subject | N-linked glycosylation | - |
dc.subject | Pichia pastoris | - |
dc.subject.mesh | Animals | en_US |
dc.subject.mesh | Cell Line, Tumor | en_US |
dc.subject.mesh | Cell Proliferation | en_US |
dc.subject.mesh | Chromatography, Affinity | en_US |
dc.subject.mesh | Cloning, Molecular | en_US |
dc.subject.mesh | Fermentation | en_US |
dc.subject.mesh | Glycosylation | en_US |
dc.subject.mesh | Humans | en_US |
dc.subject.mesh | Interleukin-3 - Genetics - Immunology - Isolation & Purification - Secretion | en_US |
dc.subject.mesh | Map Kinase Signaling System | en_US |
dc.subject.mesh | Mass Spectrometry | en_US |
dc.subject.mesh | Mice | en_US |
dc.subject.mesh | Pichia - Genetics - Metabolism | en_US |
dc.subject.mesh | Plasmids - Genetics - Metabolism | en_US |
dc.subject.mesh | Protein Sorting Signals | en_US |
dc.subject.mesh | Recombinant Fusion Proteins - Genetics - Immunology - Isolation & Purification | en_US |
dc.subject.mesh | Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization | en_US |
dc.subject.mesh | Transformation, Genetic | en_US |
dc.title | High level expression of active recombinant human interleukin-3 in Pichia pastoris | en_US |
dc.type | Article | en_US |
dc.identifier.email | Xu, A:amxu@hkucc.hku.hk | en_US |
dc.identifier.authority | Xu, A=rp00485 | en_US |
dc.description.nature | link_to_subscribed_fulltext | en_US |
dc.identifier.doi | 10.1016/j.pep.2011.08.027 | en_US |
dc.identifier.pmid | 21907288 | - |
dc.identifier.scopus | eid_2-s2.0-80052845786 | en_US |
dc.relation.references | http://www.scopus.com/mlt/select.url?eid=2-s2.0-80052845786&selection=ref&src=s&origin=recordpage | en_US |
dc.identifier.volume | 80 | en_US |
dc.identifier.issue | 2 | en_US |
dc.identifier.spage | 185 | en_US |
dc.identifier.epage | 193 | en_US |
dc.identifier.isi | WOS:000296225800004 | - |
dc.publisher.place | United States | en_US |
dc.identifier.scopusauthorid | Li, H=40261980700 | en_US |
dc.identifier.scopusauthorid | Li, N=36065390000 | en_US |
dc.identifier.scopusauthorid | Gao, X=26028577700 | en_US |
dc.identifier.scopusauthorid | Kong, X=7202794607 | en_US |
dc.identifier.scopusauthorid | Li, S=53986257400 | en_US |
dc.identifier.scopusauthorid | Xu, A=7202655409 | en_US |
dc.identifier.scopusauthorid | Jin, S=7401822323 | en_US |
dc.identifier.scopusauthorid | Wu, D=7404297751 | en_US |
dc.identifier.issnl | 1046-5928 | - |