For recombinant protein purification

cDNA corresponding to the GRAM domain of human GRAMD1b protein ({"type":"entrez-protein","attrs":{"text":"NP_065767.1","term_id":"144227752","term_text":"NP_065767.1"}}NP_065767.1) (92‐207) was cloned into the pNIC28‐Bsa4 vector with an N‐terminal His₆‐tag and a TEV‐protease cleavage site via the ligation‐independent cloning method to generate pNIC28‐Bsa4 His‐GRAMD1b_92‐207 (GRAM_1b).

The amino acid residues (K161, R189, R191, and G187) present in the GRAM_1b were mutated as indicated using site‐directed mutagenesis in pNIC28‐Bsa4 His‐GRAMD1b_92‐207 (GRAM_1b) with the following primer sets, (K161A: GRAMD1b_K161A_F and GRAMD1b_K161A_R; R189W: GRAMD1b_R189W_F and GRAMD1b_R189W_R; R191A: GRAMD1b_R191A_F and GRAMD1b_R191A_R; R189W/R191A: GRAMD1b_R189W R191A_F and GRAMD1b_R189W R191A_R; G187L: GRAMD1b_G187L_F and GRAMD1b_G187L_R), to generate pNIC28‐Bsa4 His‐GRAM_1b (K161A), pNIC28‐Bsa4 His‐GRAM_1b (R189W), pNIC28‐Bsa4 His‐GRAM_1b (R191A), pNIC28‐Bsa4 His‐GRAM_1b (R189W/R191A), and pNIC28‐Bsa4 His‐GRAM_1b (G187L), respectively. pNIC28‐Bsa4 His‐GRAM_1b (K161A) was then used as a template and mutated with the primer set GRAMD1b_R191A_F and GRAMD1b_R191A_R to generate pNIC28‐Bsa4 His‐GRAM_1b (K161A/R191A).

cDNA corresponding to the cytosolic region of human GRAMD1b ({"type":"entrez-protein","attrs":{"text":"NP_065767.1","term_id":"144227752","term_text":"NP_065767.1"}}NP_065767.1) (82‐548), including both the GRAM domain and StART‐like domain, was PCR‐amplified using EGFP‐GRAMD1b (Naito et al, 2019) as a template and the primer set 5'NcoI C‐GRAMD1b (82‐529) and 3'XhoI C‐GRAMD1b (82‐548). The PCR products were then ligated at NcoI and XhoI sites in pET28b(+) to generate pET28b(+) GRAMD1b_82‐548‐His.

The amino acid residues (K161, R189, R191, and G187) present in GRAMD1b were mutated as indicated using site‐directed mutagenesis in pET28b(+) GRAMD1b_82‐548‐His with the following primer sets, (R189W: GRAMD1b_R189W_F and GRAMD1b_R189W_R; R189W/R191A: GRAMD1b_R189W R191A_F and GRAMD1b_R189W R191A_R; R191A: GRAMD1b_R191A_F and GRAMD1b_R191A_R; G187L: GRAMD1b_G187L_F and GRAMD1b_G187L_R), to generate pET28b(+) GRAMD1b_82‐548‐His (R189W), pET28b(+) GRAMD1b_82‐548‐His (R189W/R191A), pET28b(+) GRAMD1b_82‐548‐His (R191A), and pET28b(+) GRAMD1b_82‐548‐His (G187L), respectively. pET28b(+) GRAMD1b_82‐548‐His (R191A) was then used as a template and mutated with the primer set GRAMD1b_K161A_F and GRAMD1b_K161A_R to generate pET28b(+) GRAMD1b_82‐548‐His (K161A/R191A).

gBlocks (IDT) containing either ECFP‐D4H or mVenus‐Lact‐C2 were synthesized and individually amplified by PCR, using the following primer sets (ECFP‐D4H: 5’NcoI_ECFP‐D4H and 3’BamHI_ECFP‐D4H, mVenus‐Lact‐C2: 5’NcoI_mVenus‐Lact‐C2, and 3’BamHI_pNIC28_mVenus‐Lact‐C2). The PCR products were then ligated at NcoI and BamHI sites in pNIC28‐Bsa4 to generate pNIC28‐Bsa4 His‐ECFP‐D4H and pNIC28‐Bsa4 His‐mVenus‐Lact‐C2, respectively.

cDNA corresponding to the D4H of Perfringolysin O (PFO) or the C2 domain of Lactadherin was individually PCR‐amplified using either pNIC28‐Bsa4 EGFP‐D4H or gBlock (IDT) containing EGFP‐Lact‐C2 as a template and the following primer sets (D4H: 5’NcoI_pNIC28_D4H and 3’BamHI_pNIC28_D4H; C2: 5’NheI_C2 and 3’XhoI_EGFP‐C2). The PCR products were then ligated at NcoI and BamHI sites in pNIC28‐Bsa4 for D4H and at NheI and XhoI sites for Lact‐C2 in pET28b(+) to generate pNIC28‐Bsa4 His‐D4H and pET28b(+) His‐Lact‐C2, respectively.

cDNA corresponding to the mCherry‐D4H was PCR‐amplified using mCherry‐D4H vector and the primer set D4H_F and D4H_R. The PCR products were then ligated at NcoI and MfeI sites in pNIC28‐Bsa4 to generate pNIC28‐Bsa4 mCherry‐D4H.