Generation of I-domain expression plasmids and purification of recombinant I-domains
This protocol is extracted from research article:
Exploiting species specificity to understand the tropism of a human-specific toxin
Sci Adv, Mar 11, 2020; DOI: 10.1126/sciadv.aax7515

The I-domains from human ITGAM (NM_001145808.1) and murine Itgam (NM_001082960.1), along with a 5′ Nde I site, 3′ 6-glycine linker, 3′ 3×FLAG tag, 3′ Xho I site, and the desired mutations (table S6), were produced as gBlocks Gene Fragments from Integrated DNA Technologies (IDT) and cloned into pET15b using standard cloning methods. Plasmids were transformed into E. coli T7 LysY/LacQ and purified as described previously (12). Briefly, strains were grown at 37°C, 180 rpm in LB broth supplemented with ampicillin (100 μg/ml) to an OD600 (optical density at 600 nm) of 0.5 and then induced with 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) for 3 hours. Bacteria were lysed with xTracter buffer (Clontech) supplemented with protease inhibitor (Roche), lysozyme (1 mg/ml), and Benzonase nuclease (Sigma) at 3 U/ml of culture. Lysates were incubated with nickel–nitrilotriacetic acid (NTA) resin (Qiagen), washed, and his-tagged. I-domains were eluted with 500 mM imidazole. Purified proteins were dialyzed in 1× tris-buffered saline (TBS) + 10% glycerol at 4°C overnight and then stored at −80°C.

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