pRSET A-FhFtn-1

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	The resulting plasmid construct (pRSET A-FhFtn-1) was transformed into competent E. coli BL-21 (DE3) cells (Stratagene, Santa Clara, California). Overexpression of recombinant FhFtn-1 was induced by adding isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 0.2 mM to the culture medium.	After induction, bacteria were harvested, suspended in lysis buffer (20mM sodium phosphate, 500mM NaCl, 20mM imidazole, 2% Triton X-100, 0.2mg/ml lysozyme, 1mM PMSF, pH 7.4, DNase (20µg/ml) and homogenized by sonication. Unclarified lysate was loaded onto a HisTrap FF™ crude column (GE healthcare) and washed successively with wash buffer (20mM sodium phosphate buffer pH 7.4 + 500mM NaCl + 20mM imidazole). The retained fusion protein was eluted with wash buffer (pH 5.5) containing 500mM imidazole. Eluates were desalted against wash buffer without imidazole using a PD-10 column (Amersham-Biosciences) and protein concentrations were measured using the bicinchoninic acid method [35].

Protocol tips
The resulting plasmid construct (pRSET A-FhFtn-1) was transformed into competent E. coli BL-21 (DE3) cells (Stratagene, Santa Clara, California). Overexpression of recombinant FhFtn-1 was induced by adding isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 0.2 mM to the culture medium.
Downstream tips
After induction, bacteria were harvested, suspended in lysis buffer (20mM sodium phosphate, 500mM NaCl, 20mM imidazole, 2% Triton X-100, 0.2mg/ml lysozyme, 1mM PMSF, pH 7.4, DNase (20µg/ml) and homogenized by sonication. Unclarified lysate was loaded onto a HisTrap FF™ crude column (GE healthcare) and washed successively with wash buffer (20mM sodium phosphate buffer pH 7.4 + 500mM NaCl + 20mM imidazole). The retained fusion protein was eluted with wash buffer (pH 5.5) containing 500mM imidazole. Eluates were desalted against wash buffer without imidazole using a PD-10 column (Amersham-Biosciences) and protein concentrations were measured using the bicinchoninic acid method [35].

Publication protocol

The coding region of the FhFtn-1 cDNA was amplified by PCR using a forward primer that contains a BamHI site (underlined) upstream of the start codon (5’-TGGGGATCCGCCCTTATGCACTCTGCACGC-3’, and a reverse primer that contains an EcoRI site downstream of the stop codon (5’- GGATGAATTCCAAAGTGACAATTTGCGCTCAATTC-3’). The PCR product was purified and cloned into the corresponding restriction sites of a prokaryotic expression vector pRSET A (Invitrogen) and the recombinant vector was confirmed by double-enzyme digestion and sequencing. The resulting plasmid construct (pRSET A-FhFtn-1) was transformed into competent E. coli BL-21 (DE3) cells (Stratagene, Santa Clara, California). Overexpression of recombinant FhFtn-1 was induced by adding isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 0.2 mM to the culture medium. After induction, bacteria were harvested, suspended in lysis buffer (20mM sodium phosphate, 500mM NaCl, 20mM imidazole, 2% Triton X-100, 0.2mg/ml lysozyme, 1mM PMSF, pH 7.4, DNase (20µg/ml) and homogenized by sonication. Unclarified lysate was loaded onto a HisTrap FF™ crude column (GE healthcare) and washed successively with wash buffer (20mM sodium phosphate buffer pH 7.4 + 500mM NaCl + 20mM imidazole). The retained fusion protein was eluted with wash buffer (pH 5.5) containing 500mM imidazole. Eluates were desalted against wash buffer without imidazole using a PD-10 column (Amersham-Biosciences) and protein concentrations were measured using the bicinchoninic acid method [35].

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