Expression of the soluble human Fas ligand in Dictyostelium discoideum.
- Author:
Xiao-Xia WU
1
;
Ying-Hua LU
;
Qing-Biao LI
;
Xu DENG
;
Zhi-Nan XU
Author Information
1. Department of Chemical and Biochemical Engineering, College of Chemical and Chemical Engineering, Xiamen University, Xiamen 361005, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Chorionic Gonadotropin, beta Subunit, Human;
genetics;
Culture Media;
Dictyostelium;
genetics;
growth & development;
metabolism;
Fas Ligand Protein;
biosynthesis;
genetics;
Humans;
Neutrophils;
metabolism;
Recombinant Proteins;
biosynthesis;
genetics
- From:Chinese Journal of Biotechnology
2005;21(3):380-384
- CountryChina
- Language:Chinese
-
Abstract:
An expression system is described for high-yield production of recombinant soluble human FasL (shFasL) in Dictyostelium discoideum cells. DNA encoding amino acids 141 - 281 of hFasL was PCR amplified from cDNA derived from activated human neutrophils. The resulting product was fused with a DNA fragment encoding hCG-beta signal peptide and cloned in the expression vector pMB12neo. Dictyostelium strain AX3 was transfected with this plasmid, yielding a recombinant strain called AX3-pCESFL95-H3. In order to improve the shFasL expression level, pMB12neo was optimized by replacing its transcriptional terminator/ polyadenylation segment of the 2H3 gene with an actin8 terminator/polyadenylation segment, yielding derived expression vector pMB74. The recombinant Dictyostelium strain called AX3-pLu8 was generated with this new plasmid. When the recombinant cells were cultivated in a complex HL-5C medium, a cell density of (1.5 - 2) x 10(7)/mL was reached, and the shFasL level expressed by strains AX3-pCESFL95-H3 and AX3-pLu8 was 23.5 microg/L and 206 microg/L, respectively. By using a newly developed synthetic medium called SIH as culture medium, higher cell density of (4 - 5) x 10(7)/mL was achieved. Correspondently, 111 microg/L and 420 microg/L shFasL were secreted by recombinant strains AX3-pCESFL95-H3 and AX3-pLu8, respectively.
