Expression and characterization of biologically active human Fas ligand produced in CHO cells.
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.
Zappitelli
Sabrina
S
Menarini Biotech srl, Pomezia, Roma, Italy.
D'Alatri
Laura
L
Ciucci
Allesandra
A
Raucci
Giuseppe
G
Faiella
Angela
A
Gabrielli
Meri
M
Parlani
Massimo
M
Bressan
Alessandro
A
Maggi
Carlo A
CA
Goso
Cristina
C
Rotondaro
Luigi
L
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
C505661:FASLG protein, human; D053222:Fas Ligand Protein; D008024:Ligands; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins