Figure 1
Design and purification of sCD4-17b constructs and related proteins. A) Schematic representation of three sCD4-17b constructs with different L1 linkers, with the total number of L1 amino acids indicated in the construct name (in each case consisting of the appropriate number of repeats of the G4S motif). Also shown are the constructs representing the individual components sCD4 and 17b SCFv. All constructs include the Ig kappa light chain leader sequence at the amino terminus, and the C9 epitope tag at the carboxy terminus. B) Immunoaffinity purification of sCD4-40-17b, as analyzed by Coomassie Blue staining of reducing SDS-PAGE gels (10 μl per lane for each sample). In this example, C9 peptide elution was performed in conjunction with low pH. The fractions analyzed were the initial concentrated media supernatant (load), flow-through (FT), the 5 wash fractions (W1-W5) and the two elution fractions (E1, E2). Numbers on the left indicate molecular weight markers (kDa). C) Western blot analysis of purified preparations of the indicated sCD4-17b proteins as well as the 17b SCFv and sCD4 proteins (10 μl per lane for each sample). The 1D4 MAb directed against the C-terminal tag on each protein was used for detection.