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Ncreasing concentration of PEG with a ratio 1:1. Control was performed by mixing the protein with MES buffer only with the same ratio 1:1. Murine antibody 11H6H1 [56] and FIV capsid protein p24 [57] were used as control proteins for BS3 cross-linking in the crystallization condition.Dilution experiments were conducted as described previously [57] to investigate the dissociation process of the dimer-to-monomer transition using an ITC200 calorimeter (GE Healthcare). Briefly, protein samples of FIV p15 at 6 mg/ml (375 M) into 50 mM MES pH 6 were used for sequential injections of concentrated protein solution (injection #1: 1.5 l and injections #2-16: 2.5 l), spaced at 120 seconds intervals, into the calorimetric cell (200 l), which initially contained buffer alone (50 mM MES pH 6). Measurements were performed at 25 , and data were analyzed using a dissociation model in the Origin software according to the manufacturer's instructions (GE Healthcare). The first data point was excluded in the analysis. The binding parameters H (reaction enthalpy change in cal/mol) and Kd (dissociation constant in mM) were allowed to float during the fit.Crystallization of the FIV p15 protein and data collectionSEC-MALLS experimentsTo verify the oligomeric state of the protein in solution, FIV p15 was analyzed using a SEC-MALLS (size-exclusion chromatography with multi-angle laser light scattering). We loaded 0.2 ml samples of p15 protein at 6 mg/ml in 50 mM MES pH 6, onto a Superdex 200 10/30 gelfiltration column equilibrated at 0.5 ml/min with 50 mM MES pH 6. The eluate was passed successively through a MiniDawn TREOS-angle light scattering detector (Wyatt) coupled to an Optilab T-rEX refractive index monitor (Wyatt). The data were processed, and molecular masses were calculated using the Astra V software (Wyatt).Crystallization conditions were searched using the sittingdrop vapour-diffusion method and commercial kits from Hampton Research, Molecular Dimensions Limited (MDL) and Qiagen. Crystals of FIV p15 were obtained by equilibrating drops of protein PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/4155310 (concentrated at 7 mg/ml in 50 mM MES buffer pH 6) mixed 1:1 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/12711626 with the condition 25 of the Qiagen PEGs Suite, consisting of 0.1 M sodium acetate pH 4.6 and 25 (w:v) polyethylene glycol (PEG) 3,000 at 16 . Tiny rod-shaped crystals with maximum dimension 60 ?30 ?10 m3 were tert-Butyl (7-bromoheptyl)carbamate obtained within fifteen days. Crystals were transferred to a cryoprotective solution containing the mother liquor and 10 (v:v) ethylene glycol for 30 sec and plunged into liquid nitrogen prior 5-tri-tert-butylbenzene 3-Amino-3-(4-bromophenyl)propionic acid 5-Fluoronicotinaldehyde trans-Diethyl cyclopropane-1 to data collection. First grown crystals diffracted at best to 3 ?resolution at the SLS beamline PXIII (Villigen PSI, Switzerland) and at the ESRF beamline ID23-2 (Grenoble, France) under cryo-conditions (100 K). Microseeding was necessary to optimize crystals and the best dataset was collected at 100 K to 2 ?resolution at the SOLEIL beamline 3-(2,4-Dichlorophenoxy)azetidine Proxima 1 (Paris, France). A new crystallization condition was determined for the p15-120 construct. Crystals were obtained in 0.1 M Tris pH 8, 25 (w:v) PEG 6000 (condition 45 of the Qiagen PEGs suite). Crystals were harvested as for the full-length protein and data were collected at 100 K to 2.7 ?resolution at the ESRF beamline ID29.Structure determination of FIV p15 and refinementDiffraction intensities were processed with the programs XDS [58] and XSCALE. The structure of full-length p15 was determined by the molecular replacement method using the program MrBUMP [59] of the CCP4 program suite and the structure.