SOP HPLC GFP
Geplaatst op 14 november 2005 - 20:27
Geplaatst op 14 november 2005 - 20:50
en zelfs de url van toen bestaat nu nog!!!
Green fluorescent protein was purified from E. coli strain BL21(DE3)pLysS (Novagen) containing plasmid pTu58, bearing the wild-type Aequorea victoria green fluorescent protein4. For the seleniomethionine protein, the plasmid was moved to E. coli methionine auxotroph strain B834(DE3)pLysS (Novagen). The purification involved cell lysis, centrifugation of cell debris, and four column chromatography steps: DEAE anion exchange column (Sigma, CL-6B) with a zero to 1M NaCl gradient in 10mM phosphate, 2mM EDTA, 2mM DTT, pH 7; a hydrophobic interaction column (Sigma, CL-4B) with a 0.1 to zero M phosphate gradient in 2mM EDTA, 2mM DTT, pH 7; an HPLC anion exchange column (Bio-Rad, Bio-Gel DEAE-5PW) with a zero to 1M NaCl gradient in 10 mM phosphate, 2 mM EDTA, 2mM DTT, pH 7; and an HPLC gel filtration column (Bio-Rad, Bio-Gel SEC-125) with 0.1 M phosphate, 2mM EDTA, 2mM DTT, pH 7. Gel filtration columns run at 10mM phosphate showed predominately a 2-fold higher molecular weight species. Matrix-assisted laser desorption ionization mass spectrometry was performed by the University of Texas Health Sciences Center analytical chemistry service.
The protein was crystallized in sitting drop vapor diffusion wells (Hampton Research) at room temperature using 58% 2-methyl-2,4-pentanediol (Aldrich), 50mM morpholino ethane sulfonic acid, 0.1% sodium azide at pH 6.8. The protein concentration varied, but was typically 20-30 mg/ml. Crystals grew as green fluorescent square bipyramids up to 0.5 mm on a side. The space group was determined to be P41212 or its enantiomorph, with a=b=87.15 Å and c=119.85 Å at cryogenic temperatures, and a=b= 89.23 Å and c= 119.78 Å at room temperature. The unit cell also varies with changes in ionic strength, and this effect thwarted solution by multiple isomorphous replacement. Packing density calculations suggested that there were probably two molecules per asymmetric unit.
Multi-wavelength anomalous dispersion (MAD) data were taken at Brookhaven beam line X4A at four wavelengths. The wavelengths to be used were determined by reference and crystal absorption scans. The data were taken at liquid nitrogen temperature using inverse-beam geometry in wedges of four degrees and processed using DENZO38. Native and selenio-methionine data sets were also taken in the laboratory on an R-AXIS IIC detector with CuK radiation. The native data set used in the refinement had a Rmerge of 7.7% to 1.9 Å resolution (99+% complete in all shells with 5-fold average redundancy). Selenium atoms were located initially by standard difference Patterson maps between selenium-substituted and native protein using SHELX9639 and HEAVY40 and confirmed by Patterson maps using the MAD data. MADSYS software41 was used to give the anomalous diffraction differences shown in Table 2. and to extract Fa, Ft, and phase information.
The resulting MAD-phased map was solvent flattened and two-fold averaged based on the selenium sites using CCP442, skeletonized using the program O43, and immediately revealed two 11-stranded cylindrical -barrels. The polypeptide chain was traced for one of the barrels beginning from the seleniomethionines and extending the structure in each direction, helped by the recognition of the modified tyrosine in the middle of the barrel as Tyr66, the nucleus of the fluorophore. The correct enantiomorph is space group P41212, as confirmed by the handedness of the b-barrel and the a-helices. Refinement has been started using the program X-Plor44 using the native data collected at room temperature; the current R-factor at 1.9 Å is 0.21 with an R-free of 0.26, with good geometry (rms bond and angle deviations from ideality of 0.013 Å and 1.8, respectively) and tight restraint of the non-crystallographic symmetry. All measured data were included in the refinement. Coordinates and structure factors have been deposited at the Brookhaven Protein Data Bank under accession numbers 1GFL and R1GFLSF, respectively.
Geplaatst op 14 november 2005 - 23:06
Via HPLC is maar een van de mogelijke oplossingen. Micro orange is een andere methode. Deze is echter zeer onduidelijk. Weet iemand hier toevallig iets meer van.
Geplaatst op 15 november 2005 - 11:19
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