The complementary DNA (cDNA) encoding human KV4.2 (NP_036413.1), KChIP2 (NP_775284), or DPP6 (AAI50305.1) was cloned into the pEG BacMam vector (Goehring et al., 2014 (link)). For KV4.2EM, the coding sequence was followed by a 3C protease cleavage site and a C-terminal green fluorescent protein (GFP) tag. For DPP6, a N-terminal mCherry tag was inserted before the coding sequence.
Site-directed mutagenesis were performed using Phusion or Q5 polymerase (New England Biolabs). For concatemer experiments, the cDNA encoding the second subunit was amplified, with EcoRI restriction site introduced to both ends, digested with EcoRI-HF (NEB BioLabs), and ligated into EcoRI-HF-digested, CIP (calf intestinal alkaline phosphatase, NEB BioLabs)-treated vector of the first subunit, using T4 DNA ligase (NEB BioLabs). The two subunits were connected by a SNSGGGGGGGGS linker.
For structural experiments, the expression cassette containing each gene was amplified and assembled into the pBIG1a vector using biGBac method (Weissmann et al., 2016 (link)). The multigene expression construct containing KV4.2EM/KChIP2 or KV4.2EM/KChIP2/DPP6 was used for large-scale protein expression. All constructs were verified with Sanger sequencing.
Site-directed mutagenesis were performed using Phusion or Q5 polymerase (New England Biolabs). For concatemer experiments, the cDNA encoding the second subunit was amplified, with EcoRI restriction site introduced to both ends, digested with EcoRI-HF (NEB BioLabs), and ligated into EcoRI-HF-digested, CIP (calf intestinal alkaline phosphatase, NEB BioLabs)-treated vector of the first subunit, using T4 DNA ligase (NEB BioLabs). The two subunits were connected by a SNSGGGGGGGGS linker.
For structural experiments, the expression cassette containing each gene was amplified and assembled into the pBIG1a vector using biGBac method (Weissmann et al., 2016 (link)). The multigene expression construct containing KV4.2EM/KChIP2 or KV4.2EM/KChIP2/DPP6 was used for large-scale protein expression. All constructs were verified with Sanger sequencing.