title: Expression and characterization of biologically active human Fas ligand produced in CHO cells.
abstract: We describe an expression system for high-yield production of recombinant soluble human FasL (rsh- FasL) in CHO cells. After one round of selection for gene amplification, cell lines producing rsh-FasL up to 60 microg/L x 10(6) cells in 24 h were obtained. Cell lines were grown in protein-free medium as suspension cultures. The protein secreted into growth medium was purified by immunoaffinity. The rsh-FasL thus obtained was further fractionated by gel filtration and a form of approx 140 kDa was isolated and characterized. Mass spectral analysis yielded a main peak of 28,321.15 Da, while, although to a lesser extent, dimeric and trimeric forms were also detected according to the described oligomerized state of native FasL. Our procedure permits consistent production of biologically active rsh-FasL as shown in tests on FasL-sensitive cells and in in vitro binding assays.
authors.affiliation: Menarini Biotech srl, Pomezia, Roma, Italy.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Zappitelli
authors.forename: Laura
authors.initials: L
authors.lastname: D'Alatri
authors.forename: Allesandra
authors.initials: A
authors.lastname: Ciucci
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Raucci
authors.forename: Angela
authors.initials: A
authors.lastname: Faiella
authors.forename: Meri
authors.initials: M
authors.lastname: Gabrielli
authors.forename: Massimo
authors.initials: M
authors.lastname: Parlani
authors.forename: Alessandro
authors.initials: A
authors.lastname: Bressan
authors.forename: Carlo A
authors.initials: CA
authors.lastname: Maggi
authors.forename: Cristina
authors.initials: C
authors.lastname: Goso
authors.forename: Luigi
authors.initials: L
authors.lastname: Rotondaro
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells / Q000378:metabolism* / Q000502:physiology; D002478:Cells, Cultured; D006224:Cricetinae; D053222:Fas Ligand Protein; D005784:Gene Amplification; D005786:Gene Expression Regulation; D006801:Humans; D008024:Ligands; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011786:Quality Control; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D014162:Transfection
chemical_list: C505661:FASLG protein, human; D053222:Fas Ligand Protein; D008024:Ligands; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins