Extended Data Fig. 10: Assessing the designed Ras-binding proteins with the dihydrofolate reductase (DHFR)-based protein complementarity analysis assay.
(a) The protein complementarity analysis results on 14 designed Ras-binding proteins. In these experiments, the peptide chain of DHFR is split into two parts. Ras and the protein to be assessed were separately fused with each part. Bacterium cells expressing the two fused peptides were diluted to different levels of concentrations and tittered on media containing different levels of trimethoprim (TMP), which can inhibit the endogenous DHFR activity of the cells. Possible binding between Ras and the protein to be assessed was detected through the resistance of the bacterium cells to the growth inhibition by TMP. The label ‘Raf-RBD’ represents the Ras-binding domain of Raf, which served as a positive control. The label ‘Raf-RBD R89L’ represents a mutant with abolished Ras binding activity, which served as a negative control. The stronger TMP resistance (relative to the negative control) exhibited by the cells expressing fusion peptides of the designed proteins indicated that the designed proteins examined here can bind Ras. (b) Results of competitive DHFR-PCA analysis of 4 designed proteins. In the experiments examining a designed protein, cells co-expressing isolated Raf-RBD and the DHFR-PCA system for the designed protein were analyzed. If the designed protein and Raf-RBD share binding sites on Ras, the expression of Raf-RBD, which is induced by L-arabinose, will lead to the competitive inhibition of the Ras binding of the designed protein, detected as reduced resistance to TMP.
