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Article: Large-scale production, purification and bioactivity assay of recombinant human interleukin-6 in the methylotrophic yeast Pichia pastoris

TitleLarge-scale production, purification and bioactivity assay of recombinant human interleukin-6 in the methylotrophic yeast Pichia pastoris
Authors
KeywordsExpression and purification
High-density fermentation
Interleukin-6
Pichia pastoris
Recombinant protein
Issue Date2011
PublisherBlackwell Publishing Ltd. The Journal's web site is located at http://www.blackwellpublishing.com/journal.asp?ref=1567-1356&site=1
Citation
Fems Yeast Research, 2011, v. 11 n. 2, p. 160-167 How to Cite?
AbstractA DNA fragment containing the mature human interleukin (IL)-6 sequence was cloned into pPICZαA, generating a fusion protein with the alpha factor from baker's yeast and integrated into the genome of Pichia pastoris strain X-33. Recombinant yeast transformants with high-level rhIL-6 production were identified, secreting as much as 280mgL -1 rhIL-6 after 4 days of induction by methanol. The rhIL-6 was purified by PEG-8000 precipitation, followed by DEAE anion exchange and Sephadex G-75 gel filtration, yielding over 95% pure rhIL-6 at about 170mgL -1. Mass spectrometry analysis showed that the rhIL-6 has a molecular weight of 20908.85Da, which is close to the mass calculated from the sequence of the protein. Functional analysis of the purified rhIL-6 using the lymphocyte proliferation assay by an MTT [3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl-tetrazoliumbromide] method demonstrated a specific activity that is at least fivefold higher than the commercial rhIL-6 produced in Escherichia coli. In summary, the experimental procedure we have reported here allows us to obtain a large amount of rhIL-6 from P. pastoris suitable for subsequent biophysical studies. © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.
Persistent Identifierhttp://hdl.handle.net/10722/163361
ISSN
2015 Impact Factor: 2.479
2015 SCImago Journal Rankings: 1.213
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorLi, Hen_HK
dc.contributor.authorWang, Yen_HK
dc.contributor.authorXu, Aen_HK
dc.contributor.authorLi, Sen_HK
dc.contributor.authorJin, Sen_HK
dc.contributor.authorWu, Den_HK
dc.date.accessioned2012-09-05T05:30:33Z-
dc.date.available2012-09-05T05:30:33Z-
dc.date.issued2011en_HK
dc.identifier.citationFems Yeast Research, 2011, v. 11 n. 2, p. 160-167en_HK
dc.identifier.issn1567-1356en_HK
dc.identifier.urihttp://hdl.handle.net/10722/163361-
dc.description.abstractA DNA fragment containing the mature human interleukin (IL)-6 sequence was cloned into pPICZαA, generating a fusion protein with the alpha factor from baker's yeast and integrated into the genome of Pichia pastoris strain X-33. Recombinant yeast transformants with high-level rhIL-6 production were identified, secreting as much as 280mgL -1 rhIL-6 after 4 days of induction by methanol. The rhIL-6 was purified by PEG-8000 precipitation, followed by DEAE anion exchange and Sephadex G-75 gel filtration, yielding over 95% pure rhIL-6 at about 170mgL -1. Mass spectrometry analysis showed that the rhIL-6 has a molecular weight of 20908.85Da, which is close to the mass calculated from the sequence of the protein. Functional analysis of the purified rhIL-6 using the lymphocyte proliferation assay by an MTT [3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl-tetrazoliumbromide] method demonstrated a specific activity that is at least fivefold higher than the commercial rhIL-6 produced in Escherichia coli. In summary, the experimental procedure we have reported here allows us to obtain a large amount of rhIL-6 from P. pastoris suitable for subsequent biophysical studies. © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.en_HK
dc.languageengen_US
dc.publisherBlackwell Publishing Ltd. The Journal's web site is located at http://www.blackwellpublishing.com/journal.asp?ref=1567-1356&site=1en_HK
dc.relation.ispartofFEMS Yeast Researchen_HK
dc.subjectExpression and purificationen_HK
dc.subjectHigh-density fermentationen_HK
dc.subjectInterleukin-6en_HK
dc.subjectPichia pastorisen_HK
dc.subjectRecombinant proteinen_HK
dc.subject.meshCell Proliferationen_US
dc.subject.meshChemical Fractionation - Methodsen_US
dc.subject.meshChromatography, Deae-Cellulose - Methodsen_US
dc.subject.meshChromatography, Gel - Methodsen_US
dc.subject.meshDextransen_US
dc.subject.meshGene Expressionen_US
dc.subject.meshHumansen_US
dc.subject.meshInterleukin-6 - Biosynthesis - Chemistry - Immunology - Isolation & Purificationen_US
dc.subject.meshLymphocytes - Immunologyen_US
dc.subject.meshMass Spectrometryen_US
dc.subject.meshMolecular Weighten_US
dc.subject.meshPichia - Genetics - Metabolismen_US
dc.subject.meshPolyethylene Glycols - Metabolismen_US
dc.subject.meshRecombinant Proteins - Biosynthesis - Chemistry - Immunology - Isolation & Purificationen_US
dc.subject.meshTetrazolium Salts - Metabolismen_US
dc.subject.meshThiazoles - Metabolismen_US
dc.titleLarge-scale production, purification and bioactivity assay of recombinant human interleukin-6 in the methylotrophic yeast Pichia pastorisen_HK
dc.typeArticleen_HK
dc.identifier.emailWang, Y: yuwanghk@hku.hken_HK
dc.identifier.emailXu, A: amxu@hkucc.hku.hken_HK
dc.identifier.authorityWang, Y=rp00239en_HK
dc.identifier.authorityXu, A=rp00485en_HK
dc.description.naturelink_to_subscribed_fulltexten_US
dc.identifier.doi10.1111/j.1567-1364.2010.00701.xen_HK
dc.identifier.pmid21176103-
dc.identifier.scopuseid_2-s2.0-79951481535en_HK
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-79951481535&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume11en_HK
dc.identifier.issue2en_HK
dc.identifier.spage160en_HK
dc.identifier.epage167en_HK
dc.identifier.isiWOS:000287317400002-
dc.publisher.placeUnited Kingdomen_HK
dc.identifier.scopusauthoridLi, H=39863207900en_HK
dc.identifier.scopusauthoridWang, Y=34973733700en_HK
dc.identifier.scopusauthoridXu, A=7202655409en_HK
dc.identifier.scopusauthoridLi, S=39863260500en_HK
dc.identifier.scopusauthoridJin, S=7401822323en_HK
dc.identifier.scopusauthoridWu, D=7404297751en_HK

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