Expression and Purification of mVenus Protein

The pProEx-Htb-mVenus plasmid was transformed into BL21-AI One Shot Chemically Competent E. coli cells (Thermo Fisher Scientific) and colonies grew overnight. The next morning one colony was inoculated and grown during the day. In the evening, the cells were diluted 1/100 into fresh LB with ampicillin and 1 mM IPTG and the expression proceeded overnight. The next morning the cells were pelleted and lysed with B-PER bacterial protein extraction reagent (Thermo Fisher Scientific) following the manufacture’s protocol. Then the protein was purified using TALON His-Tag Purification Resin (Clontech). The resin was washed with 300 mM NaCl/PBS and the protein was finally eluted with 250 mM imidazole in 300 mM NaCl/PBS. The protein was then concentrated using Amicon Ultra-15 centrifugal filters and the buffer was changed to PBS. Finally, the concentration of the protein was calculated based on the measured absorbance at 515 nm and extinction coefficient of 92 mM⁻¹ cm⁻¹ (ref. 40 (link)).