Purification and LPS removal of the expression of EGFPi

Purification stepaProteinEGFPiLipopolysaccharide removal
Vol (ml)Protein concnb (mg/ml)Total protein (mg)Protein/protein ratioc (μg/μg of total protein)Amt (mg)Recovery (%)Purification (fold)Concnd (EU/ml)Efficiency (fold)
    Crude lysate6724.291,627.430.03556.961001e
    After centrifugation (30,000 × g)6517.051,108.250.02123.2740.850.6>5,0001
    Organic extraction19.52.1441.730.5322.1238.8315.14NAfNA
    Triton X-114151.8427.60.6718.4932.3419.140.6>8,000
    Detoxi-Gel chromatography122.425.680.7118.233220.290.04>125,000
    S3Δ chromatography121.5180.7413.3223.3821.140.02>250,000
    Crude lysate6820.161,370.880.03345.241001e
    After centrifugation (30,000 × g)669.86650.760.01912.3627.320.58>5,0001
    Organic extraction10.52.1322.3650.5211.6325.7115.76NANA
    Triton X-11471.097.630.624.7310.4618.795>1,000
    Detoxi-Gel chromatography6.51.026.630.684.519.9720.610.07>70,000
    S3Δ chromatography6.50.483.120.712.224.9121.520.01>500,000
    Crude lysate7840.053,123.90.02990.59311001e
    After centrifugation (30,000 × g)768.65657.40.0053.2877.270.17>5,0001
    Organic extraction181.6529.70.0451.33652.951.55NANA
    Triton X-114141.2217.080.0741.263922.792.554>1,250
    Detoxi-Gel chromatography141.0414.560.0761.106562.442.620.04>125,000
    S3Δ chromatography13.51.0113.6350.081.09082.412.760.025>200,000
  • a Native EGFP (G0) and mutant EGFPi (G10 and G12) were expressed in E. coli. G10 and G12 bear single and double strands of lipopolysaccharide/lipid A binding motif of the type BHBHB, respectively (5, 6), on the β-sheet in the vicinity of the EGFP chromophore.

  • b Total protein concentration was determined by the Bradford method (1).

  • c EGFPi concentration was determined by loading a fixed amount of total protein in SDS-PAGE gels, and bands were densitometrically analyzed with Image Master VDS software version 2.0 (Amersham Pharmacia Biotech).

  • d LAL-QCL kinetic endotoxin test (BioWhittaker), the most effective and sensitive kit for detecting minute quantities (lowest limit, 0.005 EU/ml) of endotoxin in solutions, was used to quantify the efficiency of lipopolysaccharide removal.

  • e —, not detected in crude lysates because they contained soluble as well as insoluble lipopolysaccharide on the cell wall.

  • f NA, not applicable. Levels in organic extracts were not determined because they contain high salt concentrations, which may interfere with the enzymatic reaction.