Isopropyl β d 1 thiogalactoside iptg

The complete genome of Acetinobacter baumannii (ATCC 17978 strain) has been sequenced and annotated [14 (link)]. An Acinetobacter baumannii acid phosphatase gene (NCBI Accession CP000521, region: 2058753–2059719, encoding 322 amino acids) construct was synthesized with a C-terminal histidine tag, and inserted into a pET23a(+) expression vector via NdeI/HindIII restriction sites (Genscript, Inc.). The resulting pET23a-acid phosphatase (AcpA) bearing plasmid was used to transform Escherichia coli Rosetta by electroporation using a BioRad Gene Pulser Xcell apparatus. Bacterial transformants harboring pET23a-AcpA were selected on LB agar plates containing ampicillin (100 μg/ml) and chloramphenicol (50 μg/ml) followed by PCR confirmation using a pair of AcpA gene specific primers. For expression of rAcpA protein, an overnight culture of pET23a-AcpA transformant was used to inoculate 1 L LB broth while shaking at 225 RPM at 37 ⁰C to an OD₆₀₀ of 0.6. Isopropyl β-D-1-thiogalactoside (IPTG, Sigma Chemical Co.) was then added (1 mM final concentration), incubated for an additional 4 hours after which time bacterial cells were pelleted at 4000 x g for 10 minutes at 5 ⁰C. Following decanting of supernatant, the pellet was stored at -20 ⁰C.

Following protein optimization experiments, pET28a Fab′-PAS expression vector was transformed into BL21 (DE3) E. coli strain (Novagen, USA). A single colony was inoculated into 50 ml TB medium supplemented with 50 mg/ml kanamycin,1 g/lit proline, and 6 g/lit glucose, which was incubated at 37 °C overnight in a 150 rpm shaker incubator. The grown bacteria were used to inoculate 2 l TB medium supplemented with the same mentioned solutions and incubated at 30 °C in 200 rpm. The protein expression was induced with 0.5 mM isopropyl β-d-1-thiogalactoside (IPTG) (Sigma, USA) at OD_600nm 0.5 and the bacterial pellet was collected 24 h post-induction.