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| Figure 2: Characterization of sscFvPRO140. (a) Purification of sscFvPRO140. 50 ml of culture supernatant from gene-modified 293T cells expressing sscFvPRO140 was loaded onto a HisTalon cobalt column. The flow through (FT), washes (W1 and W2), and the sequential elution fractions from the same column (E1, E2, E3 and E4) were collected and equal volumes were separated by 12% SDSPAGE, followed by Coomassie blue staining. (b) Binding of sscFvPRO140 to cells expressing CCR5. 293T cells (CCR5-) or U373-MAGI-CCR5E cells (CCR5+) were incubated with purified sscFvPRO140 or PBS, followed by incubation with an anti-6xHis mAb/ HRP conjugate. OD650 was measured after addition of HRP substrate; n = 4. (c) Quantification of sscFvPRO140 in culture supernatants. 1×106 gene-modified 293T cells expressing sscFvPRO140 were grown in 2 ml of culture medium for 2 days before the culture supernatant (Sn 2) was analyzed by Western blot using an anti-6xHis mAb/HRP conjugate. Culture supernatant from cells transduced with LVX vector particles was also analyzed (Sn 1). Decreasing concentrations of purified sscFvPRO140 were analyzed in parallel to generate a protein standard curve (black bar, from left to right: 560, 280, 140, 70, and 35 nM). (d) Stability of sscFvPRO140. Culture supernatant from gene-modified 293T cells expressing sscFvPRO140 was incubated at 37°C. Aliquots were taken at the indicated time intervals and analyzed by Western blot. |
