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[Preprint]. 2023 Jul 10:2023.07.10.548208. [Version 1] doi: 10.1101/2023.07.10.548208

Extended Data Fig. 2 |. Engineering the CAGE toolbox via charge-swap mutations of the NrdJ-1 cages.

Extended Data Fig. 2 |

a, Schematic showing engineering strategy used to tune the responsiveness of the CAGE modules via charge swap mutations. The interaction between the appended caging unit and the split intein can be strengthened or weakened by manipulating key salt bridges. b, A predicted structure of the NrdJ-1 intein generated via AlphaFold2 modeling, with the amino acids for charge-swap mutations in the cages marked in green circles. c, The amino acid sequences of both NrdJ-1NCage and NrdJ-1CCage constructs (intein fragment + appended cage) with the sites altered through charge-swap mutations indicated in (b) marked in green boxes, and underlined in red. d, Schematic of MBP-GFP splice product generated by proximity-triggered CPS between complementary MBP-CAGEN and CAGEC-GFP constructs with rapalog treatment in cells. Molecular weights of the splice product and CAGE constructs are indicated. Flanking extein sequences at the splice site were either native NrdJ-1 (NPC/SEI) or AAA/SAA. e, Immunoblots of the MBP-GFP splice product (SP) bands in HEK293T cells co-expressing MBP-CAGEN and CAGEC-GFP, treated with either DMSO or rapalog (100 nM) for 4 hours. Relative splice product levels were screened with every single mutant in the cages of the CAGEN or CAGEC indicated in (b-c) and the combination of #2E/#5A in the cage of CAGEN and #1K in the cage of CAGEC. f, g, Immunoblots of the MBP-GFP splice product (SP) bands in HEK293T cells co-expressing MBP-CAGEN and CAGEC-GFP, treated with either DMSO or rapalog (100 nM) for indicated time points (f) or treated with DMSO or increasing dose of rapalog for 4 hours (g). Left – CAGE without charge-swap mutations, NPC/SEI extein sequence. Middle – engineered CAGE (#2E/#5A in the cage of CAGEN and #1K in the cage of CAGEC) NPC/SEI extein sequence. Right – engineered CAGE (#2E/#5A in the cage of CAGEN and #1K in the cage of CAGEC), AAA/SAA extein sequence. Data in (e-g) are representative of n=3 independent experiments. CAGEN refers to ‘NrdJ-1NCage-FKBP’, and CAGEC refers to ‘FRB-NrdJ-1CCage’ in (e-g). Importantly, the engineered NrdJ-1 CAGE (#2E/#5A in the cage of CAGEN and #1K in the cage of CAGEC) was found to be optimal and used in all subsequent studies.