Secretory expression and characterization of a recombinant-deleted variant of human hepatocyte growth factor in Pichia pastoris.
OBJECTIVE
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.


CONCLUSIONS
The P pastoris system represents an attractive tool of generating large quantities of hdHGF for both research and industrial purposes.
Liu
Zhi-Min
ZM
Department of Microbiological Engineering, Beijing Institute of Biotechnology, 20 Dongdajie Street, Fengtai District, Beijing 100071, China. liuzhm@vip.sina.com
Zhao
Hong-Liang
HL
Xue
Chong
C
Deng
Bing-Bing
BB
Zhang
Wei
W
Xiong
Xiang-Hua
XH
Yang
Bing-Fen
BF
Yao
Xue-Qin
XQ
D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D005285:Fermentation; D015870:Gene Expression; D014644:Genetic Variation; D017228:Hepatocyte Growth Factor / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D066298:In Vitro Techniques; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics; D010843:Pichia / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D017384:Sequence Deletion
C066855:HGF protein, human; D010446:Peptide Fragments; D011994:Recombinant Proteins; D017228:Hepatocyte Growth Factor; D004247:DNA