Figure 3

Biochemical analysis of tagged ScFv 800E6 produced in a cell-free transcription-translation system. Panel A: His(6x)-ad-N-ScFv800E6 and urokinase (UK) were transcribed-translated either in a conventional mix (disulfide -), or in a mix promoting disulfide bonding (disulfide +), as indicated, with and without Brij 35, or the chaperone DnaK. Transcription-translation was also carried out in parallel in the absence of template DNA. Supernatants (5 μl) of transcription-translation mixtures were run on a SDS-PAGE slab, side by side with two-fold dilutions of MW standards containing known amounts of a 31 kD protein, and the gel was stained by Coomassie blue. Panel B: Smaller volumes (1 μl) of the same supernatants were run as above, and electroblotted. The filter was stained with anti His-tag and anti Ig antibodies. Open and closed arrowheads mark UK and ScFv800E6 polypeptides.