Figure 3

Complementation of the tripartite split-GFP in vitro and in vivo.

(a) Complementation curves of GFP1–9 M1 (gray line) and GFP1–9 OPT (black line) with equimolar GFP10-sulfite reductase (SR)-GFP11 fusion protein (left) or GFP10–11 hairpin domain displayed on a permissive loop of a superfolder red fluorescent protein (right) (Waldo lab, manuscript under review). (b) In vivo solubility screen of 18 Pyrobaculum test proteins¹⁵ expressed in a “sandwich” configuration with N-terminal GFP10 and C-terminal GFP11 (GFP10-A-GFP11, A = protein of interest) from pTET-GFP10/11 plasmid assayed with either GFP1–10 (top) or GFP1–9 (bottom) expressed from a pET vector in BL21(DE3) E. coli.¹⁵ Fluorescence pictures of E. coli colonies on plates after 1–1/2 h Antet induction followed by IPTG induction (sequential induction) (SEQ) or 3 h co-induction (CO). Legend indicates of the tagged proteins determined by SDS-PAGE (soluble (green), partially soluble (yellow), insoluble (red)).