Site-directed mutagenesis and purification of CobB and ENO
This protocol is extracted from research article:
Protein lysine de-2-hydroxyisobutyrylation by CobB in prokaryotes
Sci Adv, Jul 17, 2019; DOI: 10.1126/sciadv.aaw6703

Using our previously reported method (3), the mutated sites of genes were introduced into the T vector–ENO–His, pET28a-CobB, and pET28a-ENO by PCR. To purify the recombinant proteins, the pET28a-CobB mutated strains and pET28a-ENO mutated strains were cultured in LB medium containing canalicillin (50 μg/ml), and cells were induced with 0.05 mM (for CobB and mutations) or 0.15 mM (for ENO and mutations) IPTG at 37°C for 4 hours. The proteins were harvested from cultured cells and purified with HisPur Ni-NTA Resin. The primers for PCR are listed in table S6.

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