Secretory expression and characterization of a recombinant-deleted variant of human hepatocyte growth factor in Pichia pastoris.
World J Gastroenterol
; 11(45): 7097-103, 2005 Dec 07.
Article
en En
| MEDLINE
| ID: mdl-16437654
AIM: To study the secretory expression of human hepatocyte growth factor (hdHGF) gene in Pichia pastoris. METHODS: The full-length gene of human cDNA encoding the deleted variant of hdHGF was cloned by RT-PCR and overlapping-fragment PCR technique using mRNA of human placenta as a template. The cloned hdHGF cDNA was inserted into the Escherichia coli-yeast shuttle vector of pPIC9. The constructed plasmid, pPIC9-hdHGF, was transformed into the GS115 cells of the methylotrophic yeast, P pastoris, using a chemical method. The Mut(+ ) transformants were screened to obtain high-expression strains by the test and analysis of expressed products of shake-flask culture. A secretory form of rhdHGF was made with the aid of the leader peptide sequence of Saccharomyces cerevisiae alpha-factor. RESULTS: The expressed products, which showed a band of molecular mass of about 80 ku, were observed on 15% SDS-PAGE and identified by Western blotting and N-terminal amino acid sequencing. In the high cell density culture of 5 L fermentor by fed-batch culture protocol, the cell biomass was reached at approximately 135 g (DCW)/L. The productivity of secreted total supernant protein concentration attained a high-level expression of more than 8.0 g/L and the ratio of rhdHGF band area was about 12.3% of the total band area scanned by SDS-PAGE analysis, which estimated that the product of rhdHGF was 500-900 mg/L. CONCLUSION: The P pastoris system represents an attractive tool of generating large quantities of hdHGF for both research and industrial purposes.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Pichia
/
Factor de Crecimiento de Hepatocito
Tipo de estudio:
Prognostic_studies
Límite:
Humans
Idioma:
En
Revista:
World J Gastroenterol
Asunto de la revista:
GASTROENTEROLOGIA
Año:
2005
Tipo del documento:
Article
País de afiliación:
China
Pais de publicación:
Estados Unidos