Fig. 7: Effect of Pro66 to Ala replacement in hDHFR on GFP-hDHFR activity following translation in Strep-Tactin-resin-assisted Ec CFS.

a Fluorescence analysis of resin-bound (RES) and free-unbound (FT) fractions of wild-type (wt) and Pro66Ala mutant (P66A) of hDHFR in GFP-hDHFR following co- and post-translational protein capture (co-/post-). Gray and black define soluble and insoluble fluorescence proportions, respectively, as described in Fig. 5b. Fluorescence units were converted to picomoles of GFP obtained from 1 ml of translation reaction as described in Fig. 5b. The results are plotted as means of two independent translation experiments. b Comparative analysis of mutant (P66A) and wild-type (wt) hDHFR in the context of GFP-hDHFR fusion protein eluted following co- or post-translational capture before (co-/post-) or after on-resin treatment of co-translationally captured protein with the buffer containing either no (NB), or 1.6 M (Gnd) of guanidine hydrochloride as described in Fig. 5c. Initial rates of fluorescence change of the respective NADPH-oxidation reactions were converted to pmol/ml of active DHFR as described in Fig. 5c. The results are plotted as means of two independent translation experiments. Source data are provided as a Source Data file.