Fig. 2
From: Lentiviral expression system for the purification of secreted proteins from human cell cultures
Utilizing alternative signal peptides to optimize protein secretion. a The signal peptide prediction software SignalP was used to analyze sCD4 with different signal peptide sequences. The C-score identifies the signal peptide cleavage site. The S-score distinguishes signal peptides from the mature protein. The Y-score is a combination of the C- and S-scores. The signal peptides, their sequence, and their final score are indicated. b 293 T cells were transduced with LV-CMV-sCD4 (CD4), LV-CMV-TPA-sCD4 (TPA), LV-CMV-AAT-sCD4 or LV-CMV-IgG-sCD4 (IgG) encoding sCD4 with the indicated signal peptide at a MOI of 2. 2 × 105 gene-modified 293 T cells were seeded in a 12-well plate in 1 ml media and cultured for 4 days. Genomic DNA was extracted from the cells and examined for the presence of integrated vector by PCR with vector-specific primers. PCR with β-actin primers was performed as a control for equal input DNA (left panel). Supernatants were collected, centrifuged, and analyzed by Western blot using an anti-His-tag antibody (left panel) and His-tag ELISA (right panel). The results are representative of 2 independent experiments performed in duplicates; *p < 0.05