pET21-EcGAPDH

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	pET21-EcGAPDH was transformed into E. coli BL21(DE3) (Novagen). A single colony from a LB–ampicillin agar plate (containing 100 mg/L ampicillin) was used to inoculate a 50 ml LB medium starter culture (containing 100 mg/L ampicillin) grown at 37 °C overnight. Then, 2 × 20 ml aliquots of the overnight culture were used to inoculate 2 × 1 L LB medium (containing 100 mg/L ampicillin). Expression was induced by the addition of 1 mM (final concentration) isopropyl-d-1-thiogalactopyranoside (IPTG) to cultures with an OD600nm of 0.6.

Protocol tips

pET21-EcGAPDH was transformed into E. coli BL21(DE3) (Novagen). A single colony from a LB–ampicillin agar plate (containing 100 mg/L ampicillin) was used to inoculate a 50 ml LB medium starter culture (containing 100 mg/L ampicillin) grown at 37 °C overnight. Then, 2 × 20 ml aliquots of the overnight culture were used to inoculate 2 × 1 L LB medium (containing 100 mg/L ampicillin). Expression was induced by the addition of 1 mM (final concentration) isopropyl-d-1-thiogalactopyranoside (IPTG) to cultures with an OD600nm of 0.6.

Publication protocol

Nde1 and Sac1 restriction sites were inserted into the 5′ and 3′ ends, respectively, of the synthesized codon optimized gapA gene open reading frame (Geneart, Carlsbad, CA, USA). The digested sequences were ligated in a pET21a plasmid previously opened with the same restriction enzyme in the cloning cassette. The resulting expression vector pET21-EcGAPDH encoded GAPDH. pET21-EcGAPDH was transformed into E. coli BL21(DE3) (Novagen). A single colony from a LB–ampicillin agar plate (containing 100 mg/L ampicillin) was used to inoculate a 50 ml LB medium starter culture (containing 100 mg/L ampicillin) grown at 37 °C overnight. Then, 2 × 20 ml aliquots of the overnight culture were used to inoculate 2 × 1 L LB medium (containing 100 mg/L ampicillin). Expression was induced by the addition of 1 mM (final concentration) isopropyl-d-1-thiogalactopyranoside (IPTG) to cultures with an OD600nm of 0.6. The cultures were maintained at 37 °C for 4 h following induction. Cells were harvested by centrifugation and resuspended in 20 mL of 50 mM Tris-HCl and 2 mM EDTA (pH 8) buffer containing 20 mM dithiothreitol (DTT). The resuspended cells were sonicated and centrifuged for 45 min at 20,000 × g. The supernatant was loaded onto a Q-Sepharose column coupled to a FPLC chromatography system (AKTA Purifer 10, GE). The protein was eluted with a salt gradient using a second buffer containing 50 mM Tris-HCl, 2 mM EDTA and 1 M KCl. The purity was checked using SDS-PAGE.

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