Expression and characterization of biologically active human fas ligand produced in CHO cells.

Autor: Zappitelli, Sabrina, D’Alatri, Laura, Ciucci, Alessandra, Raucci, Giuseppe, Faiella, Angela, Gabrielli, Meri, Parlani, Massimo, Bressan, Alessandro, Maggi, Carlo, Goso, Cristina, Rotondaro, Luigi
Zdroj: Molecular Biotechnology; Mar2003, Vol. 23 Issue 3, p189-202, 14p
Abstrakt: 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 µg/L × 106 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. [ABSTRACT FROM AUTHOR]
Databáze: Complementary Index
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