Large-scale production, purification and bioactivity assay of recombinant human interleukin-6 in the methylotrophic yeast Pichia pastoris.
FEMS Yeast Res
; 11(2): 160-7, 2011 Mar.
Article
en En
| MEDLINE
| ID: mdl-21176103
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.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Pichia
/
Expresión Génica
/
Interleucina-6
Tipo de estudio:
Prognostic_studies
Límite:
Humans
Idioma:
En
Revista:
FEMS Yeast Res
Asunto de la revista:
MICROBIOLOGIA
Año:
2011
Tipo del documento:
Article
País de afiliación:
China