pET32a- lysostaphin

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	The expression host E. coli BL21 (DE3) pLysS was used as transformation host for pET-lys vector. This strain, containing T7 RNA polymerase gene under the control of lacUV5 promoter, was transformed with pET-lys. A single colony of transformed E. coli BL21 (DE3) with pET-lys was incubated overnight on shaking incubator in 2 mL Luria-Bertani broth (LB) medium containing Ampicillin (100 μg/mL) and chloramphenicol (34 μg/mL) at 37°C with constant agitation (200 rpm). The next day, 500 μL of cultured materials was removed and inoculated in 25 mL LB broth (per liter: 14 g yeast extract,12 g Bactotryptone, 10 g NaCl, 1 g KCl, 0.5 g MgCl, 0.5 g CaCl).

Protocol tips

The expression host E. coli BL21 (DE3) pLysS was used as transformation host for pET-lys vector. This strain, containing T7 RNA polymerase gene under the control of lacUV5 promoter, was transformed with pET-lys. A single colony of transformed E. coli BL21 (DE3) with pET-lys was incubated overnight on shaking incubator in 2 mL Luria-Bertani broth (LB) medium containing Ampicillin (100 μg/mL) and chloramphenicol (34 μg/mL) at 37°C with constant agitation (200 rpm). The next day, 500 μL of cultured materials was removed and inoculated in 25 mL LB broth (per liter: 14 g yeast extract,12 g Bactotryptone, 10 g NaCl, 1 g KCl, 0.5 g MgCl, 0.5 g CaCl).

Publication protocol

"The expression host E. coli BL21 (DE3) pLysS was used as transformation host for pET-lys vector. This strain, containing T7 RNA polymerase gene under the control of lacUV5 promoter, was transformed with pET-lys. A single colony of transformed E. coli BL21 (DE3) with pET-lys was incubated overnight on shaking incubator in 2 mL Luria-Bertani broth (LB) medium containing Ampicillin (100 μg/mL) and chloramphenicol (34 μg/mL) at 37°C with constant agitation (200 rpm). The next day, 500 μL of cultured materials was removed and inoculated in 25 mL LB broth (per liter: 14 g yeast extract,12 g Bactotryptone, 10 g NaCl, 1 g KCl, 0.5 g MgCl, 0.5 g CaCl).

The culture was grown in an OD600nm of 0.6 with vigorous shaking (200 rpm) at 37°C. Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM for expression of mature lysostaphin in E. coli. The incubation period continued for another four hours at 37°C with shaking at 200 rpm. In order to produce the expression protein, bacterial suspension were tested at two and four-hour intervals and analyzed on 12% SDS-PAGE (13). The expressed protein was purified using Ni-NTA agarose column according to manufacturer’s instruction (Qiagene, Hilden, Germany).The purified protein was dialyzed and refolded with PBS (containing PMSF 0.2mM, pH = 7.2) at 4°C overnight. The quality and quantity of purified recombinant mature lysostaphin was analyzed on a 12% SDS-PAGE gel electrophoresis with Bradford methods (14)."

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