hRFVT2–pH6EX3

Protocol tips

Upstream tips	Protocol tips	Downstream tips
The E. coli optimized cDNA coding for human RFVT2 (GenBank NM NM_024531) was purchased by GenScript and subcloned in the pH6EX3 expression vector.	Chemically competent E. coli Rosetta(DE3) was transformed using the hRFVT2–pH6EX3 construct. After plate selection, colonies, carrying the recombinant plasmid, were inoculated in 100 mL of LB medium and cultured overnight at 37 °C. The pre-culture was diluted (1:10) in 1 L of fresh LB medium and incubated at 37 °C until reaching the OD of 0.6. The expression of hRFVT2 was induced adding 0.4 mM IPTG. After 4 h cells were harvested and centrifuged. The bacterial pellet was suspended in a buffer contemning NaCl 200 mM and 50 mM Hepes/Tris pH 7.5. Cells were disrupted by mild sonication at 4 °C.

Upstream tips
The E. coli optimized cDNA coding for human RFVT2 (GenBank NM NM_024531) was purchased by GenScript and subcloned in the pH6EX3 expression vector.
Protocol tips
Chemically competent E. coli Rosetta(DE3) was transformed using the hRFVT2–pH6EX3 construct. After plate selection, colonies, carrying the recombinant plasmid, were inoculated in 100 mL of LB medium and cultured overnight at 37 °C. The pre-culture was diluted (1:10) in 1 L of fresh LB medium and incubated at 37 °C until reaching the OD of 0.6. The expression of hRFVT2 was induced adding 0.4 mM IPTG. After 4 h cells were harvested and centrifuged. The bacterial pellet was suspended in a buffer contemning NaCl 200 mM and 50 mM Hepes/Tris pH 7.5. Cells were disrupted by mild sonication at 4 °C.

Publication protocol

The E. coli optimized cDNA coding for human RFVT2 (GenBank NM NM_024531) was purchased by GenScript and subcloned in the pH6EX3 expression vector. The sequence of the resulting recombinant plasmid, encoding a fusion protein corresponding to the hRFVT2 carrying an N-terminal 6-Histidine tag, was verified by sequencing. Chemically competent E. coli Rosetta(DE3) was transformed using the hRFVT2–pH6EX3 construct. After plate selection, colonies, carrying the recombinant plasmid, were inoculated in 100 mL of LB medium and cultured overnight at 37 °C. The pre-culture was diluted (1:10) in 1 L of fresh LB medium and incubated at 37 °C until reaching the OD of 0.6. The expression of hRFVT2 was induced adding 0.4 mM IPTG. After 4 h cells were harvested and centrifuged. The bacterial pellet was suspended in a buffer contemning NaCl 200 mM and 50 mM Hepes/Tris pH 7.5. Cells were disrupted by mild sonication at 4 °C.

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