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Article: High level expression of active recombinant human interleukin-3 in Pichia pastoris

TitleHigh level expression of active recombinant human interleukin-3 in Pichia pastoris
Authors
KeywordsExpression and purification
His×6-tag
Interleukin-3
N-linked glycosylation
Pichia pastoris
Issue Date2011
PublisherAcademic 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?
AbstractInterleukin-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 Identifierhttp://hdl.handle.net/10722/163399
ISSN
2021 Impact Factor: 2.025
2020 SCImago Journal Rankings: 0.458
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorLi, Hen_US
dc.contributor.authorLi, Nen_US
dc.contributor.authorGao, Xen_US
dc.contributor.authorKong, Xen_US
dc.contributor.authorLi, Sen_US
dc.contributor.authorXu, Aen_US
dc.contributor.authorJin, Sen_US
dc.contributor.authorWu, Den_US
dc.date.accessioned2012-09-05T05:30:54Z-
dc.date.available2012-09-05T05:30:54Z-
dc.date.issued2011en_US
dc.identifier.citationProtein Expression And Purification, 2011, v. 80 n. 2, p. 185-193en_US
dc.identifier.issn1046-5928en_US
dc.identifier.urihttp://hdl.handle.net/10722/163399-
dc.description.abstractInterleukin-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.languageengen_US
dc.publisherAcademic Press. The Journal's web site is located at http://www.elsevier.com/locate/yprepen_US
dc.relation.ispartofProtein Expression and Purificationen_US
dc.subjectExpression and purification-
dc.subjectHis×6-tag-
dc.subjectInterleukin-3-
dc.subjectN-linked glycosylation-
dc.subjectPichia pastoris-
dc.subject.meshAnimalsen_US
dc.subject.meshCell Line, Tumoren_US
dc.subject.meshCell Proliferationen_US
dc.subject.meshChromatography, Affinityen_US
dc.subject.meshCloning, Molecularen_US
dc.subject.meshFermentationen_US
dc.subject.meshGlycosylationen_US
dc.subject.meshHumansen_US
dc.subject.meshInterleukin-3 - Genetics - Immunology - Isolation & Purification - Secretionen_US
dc.subject.meshMap Kinase Signaling Systemen_US
dc.subject.meshMass Spectrometryen_US
dc.subject.meshMiceen_US
dc.subject.meshPichia - Genetics - Metabolismen_US
dc.subject.meshPlasmids - Genetics - Metabolismen_US
dc.subject.meshProtein Sorting Signalsen_US
dc.subject.meshRecombinant Fusion Proteins - Genetics - Immunology - Isolation & Purificationen_US
dc.subject.meshSpectrometry, Mass, Matrix-Assisted Laser Desorption-Ionizationen_US
dc.subject.meshTransformation, Geneticen_US
dc.titleHigh level expression of active recombinant human interleukin-3 in Pichia pastorisen_US
dc.typeArticleen_US
dc.identifier.emailXu, A:amxu@hkucc.hku.hken_US
dc.identifier.authorityXu, A=rp00485en_US
dc.description.naturelink_to_subscribed_fulltexten_US
dc.identifier.doi10.1016/j.pep.2011.08.027en_US
dc.identifier.pmid21907288-
dc.identifier.scopuseid_2-s2.0-80052845786en_US
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-80052845786&selection=ref&src=s&origin=recordpageen_US
dc.identifier.volume80en_US
dc.identifier.issue2en_US
dc.identifier.spage185en_US
dc.identifier.epage193en_US
dc.identifier.isiWOS:000296225800004-
dc.publisher.placeUnited Statesen_US
dc.identifier.scopusauthoridLi, H=40261980700en_US
dc.identifier.scopusauthoridLi, N=36065390000en_US
dc.identifier.scopusauthoridGao, X=26028577700en_US
dc.identifier.scopusauthoridKong, X=7202794607en_US
dc.identifier.scopusauthoridLi, S=53986257400en_US
dc.identifier.scopusauthoridXu, A=7202655409en_US
dc.identifier.scopusauthoridJin, S=7401822323en_US
dc.identifier.scopusauthoridWu, D=7404297751en_US
dc.identifier.issnl1046-5928-

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