2.1 Cloning, Expression, and Purification
The cDNA encoding Escherichia coli (strain K12) Ec ppnP,Pseudomonas aeruginosa (strain PA0750) Pa ppnP,Vibrio cholerae (strain SO5Y) Vc ppnP, Salmonella
enterica (subsp. enterica serovar Typhimurium strain SL7207)St ppnP are ordered from Tsingke Biotechnology Co., Ltd. All the
coding sequences of these genes are inserted into a modified pET-28a
vector with 6xHis-Sumo tag and a ULP1 protease site. The vectors are
transformed into E. coli BL21(DE3 ) competent cells and
coated plates with Kana antibiotic. After incubation overnight, a single
colon was picked to the LB medium supplied with 50 mg/L kanamycin. TheE. coli cells were cultured in LB medium at 37℃ until the
OD600 reached 0.6-0.8, then the bacteria were induced
with 0.2 mM IPTG at 18℃ for 12 h. The culture was collected by
centrifugation, resuspended in buffer containing 20 mM Tris HCl pH8.0,
500 mM NaCl, 20 mM imidazole pH 8.0, and lysed by high pressure. Cell
extracts were centrifuged at 17,000 rpm for 1h at 4 ℃. Supernatants were
purified with Ni-NTA (GE), the target protein was washed with lysis
buffer, and then eluted with a buffer containing 20mM Tris-HCl, pH8.0,
500mM NaCl, and 500mM imidazole. Ulp1 protease was added to remove the
N-terminal tag and fusion protein of the recombinant protein and
dialyzed with lysis buffer for 3 hours. The mixture was applied to
another Ni-NTA resin to remove the protease and uncleaved proteins.
Eluted proteins were concentrated by centrifugal ultrafiltration, loaded
onto a pre-equilibrated HiLoad 16/60 Superdex 75-pg column (GE
Healthcare), eluted at a flow rate of 1 ml/min with the same buffer
containing 10mM Tris-HCl pH8.0, 100 mM NaCl. Peak fractions were
analyzed by SDS-PAGE (15%, w/v) and stained with Coomassie Brilliant
Blue R-250. Purified fractions were pooled together and concentrated by
centrifugal ultrafiltration. The concentration was determined by A280.
The protein was concentrated to 10 mg/ml for crystallization trials.