GFP-like sequences were predicted from Assembly 1 of the amphioxus genome  and gene predictions, using Blast. Alleles were distinguished by inspection of intronic sequence conservation, mapping to Assembly 2, and identity of neighboring genes. Ka/Ks ratios were calculated using DnaSp  and an online server . Searches for GFPs in other sequence sources used Blast, Psi-Blast, HMMer and Gene Detective, a hardware implementation of the Genewise algorithm (Active Motif, Carlsbad, CA). ESTs were downloaded from NCBI on Nov 12, 2008, and Blasted against GFP nucleotide sequences. Library information was extracted from EST FastA headers.
RNA Extraction and Protein Cloning
Branchiostoma floridae (amphioxus), collected from Tampa Bay, Florida were frozen at -80°C, ground to a paste using a mortar and pestle and RNA extracted using TriReagent (Sigma) and the RNeasy kit (Invitrogen, Carlsbad, California) according to the manufacturer's protocol. cDNA was made with the Retroscript kit (Ambion, Austin, Texas). Gene-specific primers for GFPa1, GFPc1, GFPd2, and GFPe1 were designed based on sequences from the genome database (JGI) and used to amplify GFP-like genes from amphioxus cDNA by polymerase chain reaction (PCR). Gene-specific primers for GFPf1 were designed based on sequences from the genome database (JGI) and used to amplify GFPf1 from amphioxus EST clone CAXG1077. PCR products were cloned into either the pET24b(+) E. coli expression vector (Novogen, New Canaan, Connecticut) at NdeI and HindIII restrictions sites or the pHIS8 E. coli expression vector at NcoI and HindIII restrictions sites .
Protein Expression and Purification
Transformed E. coli BL21(DE3) cells were incubated with shaking at 37°C in Luria broth  containing 50 μg/ml kanamycin until OD600 nm = 0.8. Protein expression was induced with 0.5 mM isopropyl 1-thio-β-galactopyranoside (IPTG) and the cultures were incubated with shaking at 37°C for 4 hours. Cells were harvested by centrifugation at 9,000 g and cell pellets resuspended in lysis buffer [400 mM NaCl, 50 mM Tris-HCl (pH 8.0), 10% glycerol, 10 mM BME] supplemented with 0.5 mg/ml lysozyme. Following sonication and centrifugation at 100,000 g, supernatant was passed over a Ni2+-NTA column (Qiagen, Valencia, CA) equilibrated in lysis buffer, washed with 10 bed volumes of wash buffer [400 mM NaCl, 50 mM Tris-HCl (pH 8.0), 20 mM imidazole, 10 mM BME] and the His-tagged protein eluted with 10 bed volumes of elution buffer [400 mM NaCl, 50 mM Tris-HCl (pH 8.0), 250 mM imidazole, 10 mM BME]. Subsequently, 30 units of thrombin protease were added and the eluted protein was dialyzed overnight against 400 mM NaCl, 50 mM Tris pH8, 10 mM BME. The retentate was reloaded onto a Ni2+-NTA column to remove the protein bearing uncleaved His-tags and then passed over a benzamidine sepharose column to remove the thrombin protease. The resulting flowthrough was concentrated and loaded onto a Superdex S200 gel filtration column (Amersham, Piscataway, New Jersey) equilibrated in gel filtration buffer [400 mM NaCl 50 mM TRIS-HCl (pH 8.0), 1 mM DTT] to isolate homogeneous dimeric GFPa1, GFPc1, GFPd2, GFPe1, and GFPf1. Peak fractions were collected, concentrated, and stored at -80°C.
Fluorescence spectra were recorded with a SE200 low-light digital spectrograph (Catalina Scientific Instruments, Tucson, AZ) upon excitation with a 465 nm LED (Ocean Optics, Dunedin, FL) that showed a narrow spectrum as defined by the Full Width at Half the Maximum intensity (FWHM = 31 nm). To measure fluorescence spectra GFP-like proteins were diluted to 1 μM and excitation was set at 800 msec and zero electronic gain. All dilutions were done with gel filtration buffer. Normalized fluorescence spectra were measured with proteins diluted to 1 μM (GFPa1) or 10 μM (GFPc1, GFPd2, GFPe1, and GFPf1). Excitation was set at 800 msec and zero electronic gain (GFPa1) or 5 sec with 150 electronic gain (GFPe1, GFPc1, GFPd2, and GFPf1). Absorbance spectra were recorded with the same apparatus. Normalized absorbance spectra were recorded from proteins diluted to 10 μM. A Spectra Max M2 (Molecular Devices, Sunnyvale, CA) spectrophotometer was used to measure excitation spectra at 10 μM protein concentration. Fluorescence emission was set at 515 nm while excitation was scanned from 350–505 nm.
Protein concentrations were calculated using the extinction coefficient of the chromophore after denaturation in 0.1 N NaOH (44, 000 M-1 cm-1 at 446 nm) [41, 42].
Absorbance of GFPa1, GFPc1, GFPd2, GFPe1, and GFPf1 was measured using a Spectra Max M2 (Molecular Devices, Sunnyvale, CA) spectrophotometer and extinction coefficients calculated according to the Beer Lambert law.
bfloGFP Antioxidant Capacity
The total antioxidant status assay (EMD Biosciences, Darmstadt, Germany) was used to evaluate antioxidant activity. This assay is based upon the inhibition of the ferrylmyoglobin•-catalyzed oxidation of colorless ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) into ABTS• which is green in color and absorbs at 600 nm. Addition of antioxidant quenches the ferrylmyoglobin•, inhibits ABTS• formation, and decreases absorbance at 600 nm. GFPs do not absorb or fluoresce in the 600 nm range, and so they do not optically interfere in this assay. Total antioxidant status assay reactions were carried out according to manufacturer's protocol. Briefly, 20 μL of either sample buffer, a protein sample, or the reaction standard, 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, was added to 1 ml of phosphate buffered saline containing metmyoglobin and ABTS and vortexed. 200 μL of H2O2 substrate was added, vortexed, and the absorbance at 600 nm was followed for 3 minutes. All proteins were diluted to 200 μM prior to addition. Addition of GFP buffer alone (50 mM Tris pH 8.0, 400 mM NaCl, 1 mM DTT) served as a control reaction while addition of lysozyme (Sigma, St. Louis, Missouri) served as a negative control. Absorbance values were expressed relative to the reaction standard. The assay measurements were completed three independent times (N = 3). At the end of the assay, OD values from each sample measurement were expressed relative to the mean OD obtained from the standard. Relative OD values were then log(x+1) transformed to respect homocedasticity of the statistical analysis (Zar, 1996). Differences among samples were tested for statistical significance with α of 0.05 for two-tailed comparisons. Analysis of Variance (ANOVA) and post-hoc multiple comparison of means (Fisher's PLSD) were used to test differences, using Statview 5.0 (SAS Institute, Inc.).