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- Publisher Website: 10.1111/j.1567-1364.2010.00701.x
- Scopus: eid_2-s2.0-79951481535
- PMID: 21176103
- WOS: WOS:000287317400002
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Article: Large-scale production, purification and bioactivity assay of recombinant human interleukin-6 in the methylotrophic yeast Pichia pastoris
Title | Large-scale production, purification and bioactivity assay of recombinant human interleukin-6 in the methylotrophic yeast Pichia pastoris |
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Authors | |
Keywords | Expression and purification High-density fermentation Interleukin-6 Pichia pastoris Recombinant protein |
Issue Date | 2011 |
Publisher | Blackwell 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? |
Abstract | A 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 Identifier | http://hdl.handle.net/10722/163361 |
ISSN | 2023 Impact Factor: 2.4 2023 SCImago Journal Rankings: 0.630 |
ISI Accession Number ID | |
References |
DC Field | Value | Language |
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dc.contributor.author | Li, H | en_HK |
dc.contributor.author | Wang, Y | en_HK |
dc.contributor.author | Xu, A | en_HK |
dc.contributor.author | Li, S | en_HK |
dc.contributor.author | Jin, S | en_HK |
dc.contributor.author | Wu, D | en_HK |
dc.date.accessioned | 2012-09-05T05:30:33Z | - |
dc.date.available | 2012-09-05T05:30:33Z | - |
dc.date.issued | 2011 | en_HK |
dc.identifier.citation | Fems Yeast Research, 2011, v. 11 n. 2, p. 160-167 | en_HK |
dc.identifier.issn | 1567-1356 | en_HK |
dc.identifier.uri | http://hdl.handle.net/10722/163361 | - |
dc.description.abstract | A 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.language | eng | en_US |
dc.publisher | Blackwell Publishing Ltd. The Journal's web site is located at http://www.blackwellpublishing.com/journal.asp?ref=1567-1356&site=1 | en_HK |
dc.relation.ispartof | FEMS Yeast Research | en_HK |
dc.subject | Expression and purification | en_HK |
dc.subject | High-density fermentation | en_HK |
dc.subject | Interleukin-6 | en_HK |
dc.subject | Pichia pastoris | en_HK |
dc.subject | Recombinant protein | en_HK |
dc.subject.mesh | Cell Proliferation | en_US |
dc.subject.mesh | Chemical Fractionation - Methods | en_US |
dc.subject.mesh | Chromatography, Deae-Cellulose - Methods | en_US |
dc.subject.mesh | Chromatography, Gel - Methods | en_US |
dc.subject.mesh | Dextrans | en_US |
dc.subject.mesh | Gene Expression | en_US |
dc.subject.mesh | Humans | en_US |
dc.subject.mesh | Interleukin-6 - Biosynthesis - Chemistry - Immunology - Isolation & Purification | en_US |
dc.subject.mesh | Lymphocytes - Immunology | en_US |
dc.subject.mesh | Mass Spectrometry | en_US |
dc.subject.mesh | Molecular Weight | en_US |
dc.subject.mesh | Pichia - Genetics - Metabolism | en_US |
dc.subject.mesh | Polyethylene Glycols - Metabolism | en_US |
dc.subject.mesh | Recombinant Proteins - Biosynthesis - Chemistry - Immunology - Isolation & Purification | en_US |
dc.subject.mesh | Tetrazolium Salts - Metabolism | en_US |
dc.subject.mesh | Thiazoles - Metabolism | en_US |
dc.title | Large-scale production, purification and bioactivity assay of recombinant human interleukin-6 in the methylotrophic yeast Pichia pastoris | en_HK |
dc.type | Article | en_HK |
dc.identifier.email | Wang, Y: yuwanghk@hku.hk | en_HK |
dc.identifier.email | Xu, A: amxu@hkucc.hku.hk | en_HK |
dc.identifier.authority | Wang, Y=rp00239 | en_HK |
dc.identifier.authority | Xu, A=rp00485 | en_HK |
dc.description.nature | link_to_subscribed_fulltext | en_US |
dc.identifier.doi | 10.1111/j.1567-1364.2010.00701.x | en_HK |
dc.identifier.pmid | 21176103 | - |
dc.identifier.scopus | eid_2-s2.0-79951481535 | en_HK |
dc.relation.references | http://www.scopus.com/mlt/select.url?eid=2-s2.0-79951481535&selection=ref&src=s&origin=recordpage | en_HK |
dc.identifier.volume | 11 | en_HK |
dc.identifier.issue | 2 | en_HK |
dc.identifier.spage | 160 | en_HK |
dc.identifier.epage | 167 | en_HK |
dc.identifier.isi | WOS:000287317400002 | - |
dc.publisher.place | United Kingdom | en_HK |
dc.identifier.scopusauthorid | Li, H=39863207900 | en_HK |
dc.identifier.scopusauthorid | Wang, Y=34973733700 | en_HK |
dc.identifier.scopusauthorid | Xu, A=7202655409 | en_HK |
dc.identifier.scopusauthorid | Li, S=39863260500 | en_HK |
dc.identifier.scopusauthorid | Jin, S=7401822323 | en_HK |
dc.identifier.scopusauthorid | Wu, D=7404297751 | en_HK |
dc.identifier.issnl | 1567-1356 | - |