The rpoC gene (RpoC, accession no.:
NP_268496.1) coding for N-terminal residues Phe7-Asp818 was amplified
by polymerase chain reaction (PCR) from the genomic DNA of the S. pyogenes M1 strain (MTCC) and cloned in the pEC-K-HT-HIS
(2) in-house vector at EcoR1 and BamH1 sites. The construct was transformed
into the expression host BL21 (DE3) strain of Escherichia
coli. For protein expression, the transformed E.
coli cells were cultured in Luria–Bertani (LB) medium.
An overnight culture (10 mL) was prepared and transferred to 1 L of
LB medium supplemented with 50 mg/mL kanamycin. The culture was grown
at 37 °C at 150 rpm shaking until the desired optical density
of 0.6 at A600 was obtained. The culture was induced with
1 mM isopropyl β-d -1-thiogalactopyranoside (IPTG) for
protein expression and incubated further for 4 h with shaking. The
cells were harvested by centrifugation at 4000 rpm for 20 min at 4
°C. The cell pellet was suspended in lysis buffer containing
20 mM Tris pH 7.0, 300 mM NaCl, and 10% glycerol, and the cells were
lysed by sonication on ice. The cell lysate was centrifuged at 10,000
rpm for 45 min at 4 °C, and the pellet was used for purification.
The protein was solubilized from the pellet using buffer containing
20 mM Tris pH 7.0, 300 mM NaCl, 10% glycerol, and 10% N-lauroylsarcosine by continuous stirring at 150 rpm for 1 h at 4
°C and centrifuged at 10,000 rpm for 30 min at 4 °C. The
solubilized protein was further refolded by stepwise membrane dialysis
against lysis buffer containing 5, 2.5, 1, and 0% N-lauroylsarcosine, respectively, for 3 h at 4 °C. As the protein
was expressed with an N-terminal His-tag, purification was carried
out by nickel-affinity chromatography. At each step, the purity of
the fractions was analyzed on a 10% SDS-PAGE gel. The concentration
of protein was measured using a UV spectrophotometer (A280) and presumed calculated absorption coefficient of 0.789 from the
PROTPARAM online tool.29
NP_268496.1) coding for N-terminal residues Phe7-Asp818 was amplified
by polymerase chain reaction (PCR) from the genomic DNA of the S. pyogenes M1 strain (MTCC) and cloned in the pEC-K-HT-HIS
(2) in-house vector at EcoR1 and BamH1 sites. The construct was transformed
into the expression host BL21 (DE3) strain of Escherichia
coli. For protein expression, the transformed E.
coli cells were cultured in Luria–Bertani (LB) medium.
An overnight culture (10 mL) was prepared and transferred to 1 L of
LB medium supplemented with 50 mg/mL kanamycin. The culture was grown
at 37 °C at 150 rpm shaking until the desired optical density
of 0.6 at A600 was obtained. The culture was induced with
1 mM isopropyl β-
protein expression and incubated further for 4 h with shaking. The
cells were harvested by centrifugation at 4000 rpm for 20 min at 4
°C. The cell pellet was suspended in lysis buffer containing
20 mM Tris pH 7.0, 300 mM NaCl, and 10% glycerol, and the cells were
lysed by sonication on ice. The cell lysate was centrifuged at 10,000
rpm for 45 min at 4 °C, and the pellet was used for purification.
The protein was solubilized from the pellet using buffer containing
20 mM Tris pH 7.0, 300 mM NaCl, 10% glycerol, and 10% N-lauroylsarcosine by continuous stirring at 150 rpm for 1 h at 4
°C and centrifuged at 10,000 rpm for 30 min at 4 °C. The
solubilized protein was further refolded by stepwise membrane dialysis
against lysis buffer containing 5, 2.5, 1, and 0% N-lauroylsarcosine, respectively, for 3 h at 4 °C. As the protein
was expressed with an N-terminal His-tag, purification was carried
out by nickel-affinity chromatography. At each step, the purity of
the fractions was analyzed on a 10% SDS-PAGE gel. The concentration
of protein was measured using a UV spectrophotometer (A280) and presumed calculated absorption coefficient of 0.789 from the
PROTPARAM online tool.29
