Gateway recombinational cloning

Full-length wildtype (WT) fpn-1.1 with C-terminal FLAG tag was PCR amplified using primers 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTACATGGCTTGGTTATCCGGAAAAG-3′ and 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTTTCACTTGTCATCGTCGTCCTTGTAGTCTTCAAAAGTTGGCGAATCCAAC-3′ from cDNA library which was converted from total RNAs isolated from N2 worms (see below). The plasmid was created with Gateway recombinational cloning (Invitrogen). The above PCR product was initially recombined with the pDONR221 vector to create the pENTRY clone. Next, the fpn-1.1 pENTRY construct was recombined into pDEST-sur-5 vector^{25 (link)}, under the promoter of the acetoacetyl-coenzyme A synthetase (sur-5) gene. This plasmid was then used to create transgenic worms.

Wild-type (WT) and mutant human BRAF (232–284) and CRAF/RAF-1 (136–188) entry clones were synthesized with an upstream tobacco etch virus (TEV) protease cleavage site (ENLYFQ/G) and optimized for expression in E. coli by ATUM (Newark, CA). Entry clone inserts were transferred by Gateway recombinational cloning (ThermoFisher, Carlsbad, CA) to pDest-566 (Addgene #11517), a T7-based E. coli expression vector encoding an N-terminal His6-MBP (maltose-binding protein) fusion. All CRD proteins were expressed using LB medium supplemented with 300 mM ZnCl₂ as outlined for CRAF (52–188) in (Lakshman et al., 2019 (link)). For 2-liter working volume fermentations, 3-liter Bioflo 110 (Eppendorf) were used, and 20-liter Bioflo IV (Eppendorf) were used for 15-liter working volumes. Antifoam 204 (Sigma) was added at 0.075% v/v and 0.017% v/v, respectively, to the 2- and 15-liter fermenters. All proteins were purified essentially as outlined in (Kopra et al., 2020 (link)) for CRAF RBD (52–131). Specifically, 5 mM TCEP was used in all buffers and the protein occasionally eluted overlapping the salt peak during SEC which then required an additional dialysis step (3K MWCO) to the final buffer.

Gateway recombinational cloning (Thermo Fisher Scientific) was used to make an untagged rhinovirus 3C protease expression vector for co-lysis experiments. The 3C protease ORF was amplified by PCR from pET/3C using primers PE-2856 and PE-2857 (Table 1). These primers incorporated the attB recombination sites along with an appropriately positioned ribosome-binding site (RBS) in the PCR product. Subsequently, the PCR product was used in a BP reaction with pDONR221 (Thermo Fisher Scientific) to generate the entry clone pSRK2703. The entry clone was recombined in an LR reaction with pDEST42 (Thermo Fisher Scientific) to generate the IPTG-inducible 3C protease expression vector pSRK2706 (Table 2).