title: Large-scale production, purification and bioactivity assay of recombinant human interleukin-6 in the methylotrophic yeast Pichia pastoris.
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 280 mg L(-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 170 mg L(-1) . Mass spectrometry analysis showed that the rhIL-6 has a molecular weight of 20,908.85 Da, 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.
authors.affiliation: The Key Laboratory of Regenerative Biology, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.
authors.forename: Hongbo
authors.initials: H
authors.lastname: Li
authors.forename: Yu
authors.initials: Y
authors.lastname: Wang
authors.forename: Aimin
authors.initials: A
authors.lastname: Xu
authors.forename: Shiwu
authors.initials: S
authors.lastname: Li
authors.forename: Shouguang
authors.initials: S
authors.lastname: Jin
authors.forename: Donghai
authors.initials: D
authors.lastname: Wu
mesh_terms: D049109:Cell Proliferation; D005591:Chemical Fractionation / Q000379:methods; D002848:Chromatography, DEAE-Cellulose / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D003911:Dextrans; D015870:Gene Expression; D006801:Humans; D015850:Interleukin-6 / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification*; D008214:Lymphocytes / Q000276:immunology; D013058:Mass Spectrometry; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics* / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D013778:Tetrazolium Salts / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism
chemical_list: D003911:Dextrans; D015850:Interleukin-6; D011994:Recombinant Proteins; D013778:Tetrazolium Salts; D013844:Thiazoles; D011092:Polyethylene Glycols; C025614:sephadex; C022616:thiazolyl blue