One shot bl21 de3 chemically competent e coli

The duck LEAP-2/pCR2.1-TOPO vector was digested with the restriction enzymes EcoRI and BamHI (Promega, Madison, WI, USA). The protein expression vector pET32a (Novagen, Madison, WI, USA) was also digested with the same restriction enzymes. The digested fragments were purified from the agarose gel using the PureLink Quick Gel Extraction Kit (Invitrogen, USA) and were ligated using T4 DNA Ligase (Invitrogen, USA). The ligated vector and insert were transformed into One Shot BL21 (DE3) Chemically Competent E. coli (Invitrogen, USA) and sequenced. Positive clones were incubated at 37°C overnight on a shaking incubator at 225 rpm in LB broth with ampicillin (50 μg/mL). The bacteria culture was then induced for recombinant protein expression with 1 mM isopropyl-β-D-thiogalctopyranoside (USB Corporation, Cleveland, OH, USA) for 4 h at 28°C, and the bacteria were centrifuged at 5,000×g for 15 min. The duck LEAP-2 recombinant protein was extracted with B-PER Bacterial Protein Extraction Reagent (Thermo Scientific, USA) and purified using HisPur Cobalt Resin (Thermo Scientific, USA). Recombinant duck LEAP-2 was eluted using 250 mM imidazole and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting using 6× His-tag antibody (Thermo Scientific, USA).

BamHI or NdeI-ended coding sequences for the selected antigens were amplified from S. pyogenes gDNA (strain H305) and cloned into pET-19b excluding any signal sequence and cell wall anchor domain (Supplementary Table 1). For pulA (Spy_1972) a 1.6 kb N-terminal fragment was amplified by PCR and cloned into the TA cloning vector PCR 2.1 (Invitrogen) to facilitate NdeI digestion. For scpA, a premature stop codon was inserted at amino acid 720 by site directed mutagenesis (QuikChange II XL, Agilent) to prevent removal of the N-terminal propeptide.¹⁵ (link) Antigens were produced by IPTG-induced overexpression in One Shot^® BL21 (DE3) Chemically Competent E. coli (Invitrogen) according to the manufacturer's instructions. The induced BL21 cells were lysed with BugBuster (Novagen) and proteins were purified using the Ni-NTA purification system (Invitrogen) according to the manufacturer's instructions.

The expression plasmids were transformed into One-Shot™ BL21(DE3) chemically competent E. coli (Invitrogen) according to the manufacturer’s instructions. The transformation reaction was plated into LB agar plates containing 0.1 mg/ml ampicillin. After incubation overnight at 37°C, a colony was transferred into 100 ml of MagicMedia™ E. coli expression medium (Thermo Fisher Scientific). The medium was then incubated at 30°C for 20 h on a shaker (220 rpm).
The bacterial cells were harvested via centrifugation (3,000 g, RT, 10 min), washed once with 20 mM of sodium phosphate (pH 7.4), and frozen at −20°C. The recombinant, his-tagged protein was purified using a Protino Ni-TED 1000 Kit (Machery-Nagel, Oensingen, Switzerland) according to the manufacturer’s protocol. The buffer of the eluate containing the recombinant protein was exchanged with 20 mM of sodium phosphate (pH 7.4) using illustra™ NAP™ columns (VWR International GmbH, Dietikon, Switzerland) according to the manufacturer’s protocol. The protein concentration was determined using the Qubit Protein Assay Kit (Thermo Fisher Scientific). The purity of the purified proteins was evaluated using denaturing polyacrylamide gel electrophoresis, followed by Coomassie Blue staining.