Cyan Fluorescent Protein Carries a Constitutive Mutation That Prevents Its Dimerization (original) (raw)

Expression and purification of fluorescent proteins, His-tag cleavage. Competent E. coli cells (Top10 or BL21 strain) were transformed with pHis-CFP, pHis-CFP A206K, pHis-CFP I146N or pHis-YFP. A volume of 1.5 L of Terrific Broth medium (TB) containing 100 μg/mL ampicillin was inoculated with a 50 mL starter culture that had been grown overnight. Protein production was induced at OD(600 nm) = 0.6-0.7 using 1 mM isopropyl-β-D-thio-galactopyranoside (IPTG). After 18 h of culture at 30°C, bacteria were harvested by centrifugation and frozen. The cells were resuspended in 25 mL of lysis buffer (50 mM Tris-HCl, 5 mM 2-mercaptoethanol, 1 mM PMSF, 0.02 mg/mL DNase), and sonicated. The cell debris were removed by centrifugation at 40000 rpm for 1h30 at 6°C. The supernatant was filtered with a 0.22 μm filter and diluted by a factor 2 with phosphate buffer (30 mM NaH 2 PO 4 , 700 mM NaCl, 30 mM imidazole, pH 7.5). The solution was then applied to a column containing 15 mL of Ni-NTA agarose (Sigma) for 1h. The column was then washed (30 mM NaH 2 PO 4 , 100 mM NaCl, 10 mM imidazole, pH 7.5) and the protein was eluted (30 mM NaH 2 PO 4 , 100 mM NaCl, 150 mM imidazole, pH 7.5). Purity was checked by SDS-PAGE. At this stage, the His-tag was possibly cleaved following the protocol described in [1]. Before storage at-20°C, the protein solutions were dialyzed against phosphate buffer (30 mM NaH 2 PO 4 , pH 7.4) or PBS (4.3 mM Na 2 HPO 4 , 1.5 mM KH 2 PO 4 , 137 mM NaCl, 2.7 mM KCl, pH 7.4) for further experiments. Molar extinction coefficients. Steady-state absorption spectra were acquired on a Perkin Elmer Lambda 900 spectrophotometer, in 3 x 3 mm cuvettes. The molar extinction coefficient of each protein was obtained using Beer's law. The absorbance of several dilutions of the protein stock solution was measured, and their concentration was determined independently by the BCA assay. [2] We found ε = 30500 M-1 .cm-1 for CFP, ε = 27000 M-1 .cm-1 for CFP A206K and ε = 26000 M-1 .cm-1 for CFP I146N, at the absorption maximum. These values are not significantly different and agree well with the literature. [3-5] In binding titration experiments, protein concentrations were calculated based on an average extinction coefficient of 28000 M-1 .cm-1 for all CFPs. YFP samples for sedimentation velocity measurements were prepared by dilution of the stock solution, which was titrated using the BCA assay. [2] Corrected excitation and emission spectra of fluorescent proteins. Fluorescence excitation and emission spectra were measured on a Jobin Yvon Fluorolog 3 fluorimeter at 20°C in 3 x 3 mm cuvettes on dilute solutions (OD ≈ 0.05). Excitation spectra were subtracted from the buffer signal and corrected from the lamp spectrum using the correction file supplied by Jobin Yvon. For emission spectra, fluorescence was collected at magic angle (54.7°) with respect to the excitation, to avoid polarization artifacts. As a matter of fact, the rotational correlation time of GFPs (16 ns [6]) is larger than the excited-state lifetime (a few ns, depending on the fluorescent protein), so that the fluorescence emitted by fluorescent proteins is significantly polarized. The emission spectra were