Recombinant Human CD5L Production

The cDNA of human CD5L (hsCD5L) was obtained by gene synthesis (GenScript) following NCBI reference sequence NP_005885.1, with a modification in which the immunoglobulin g chain signal peptide replaced that of CD5L. The cDNA was cloned into the p.evi vector and transiently transfected into CHO K1 cells using the eviFect system (Evitria AG). Cells were grown in eviMake, a chemically defined, serum-free, animal component-free medium. The cell culture supernatant fraction was harvested at d 8 after transfection, dialyzed to 20 mM Na₂HPO₄, pH 7.4 and subjected to MonoQ chromatography. Recombinant human CD5L (r-HsCD5L) was eluted in a sodium chloride gradient, and purification was monitored by SDS-PAGE. Purified protein was dialyzed to PBS, concentrated by centrifugation on Amicon ultra (Millipore, UFC901024), and possible endotoxin contamination was removed by Endotrap columns (Hyglos GmbH, 321063), following the manufacturer´s protocol and as performed before.⁵ (link) As mentioned above, the purified r-HsCD5L was tested in preliminary experiments, where its activity in terms of MФ TNF secretion, as well as inhibition of apoptosis induced by cycloheximide treatment, was comparable to that of commercially available r-HsCD5L (R&D Systems) (data not shown).