[Expression of the soluble human Fas ligand in Dictyostelium discoideum].
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.
Wu
Xiao-Xia
XX
Department of Chemical and Biochemical Engineering, College of Chemical and Chemical Engineering, Xiamen University, Xiamen 361005, China.
Lu
Ying-Hua
YH
Li
Qing-Biao
QB
Deng
Xu
X
Xu
Zhi-Nan
ZN
D000818:Animals; D018997:Chorionic Gonadotropin, beta Subunit, Human / Q000235:genetics; D003470:Culture Media; D004023:Dictyostelium / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D053222:Fas Ligand Protein / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D009504:Neutrophils / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D018997:Chorionic Gonadotropin, beta Subunit, Human; D003470:Culture Media; D053222:Fas Ligand Protein; D011994:Recombinant Proteins