This predicted -strand might play a role similar to that of the vIL-6 -strand, located in the middle of a larger ACB loop and consisting of the IFHLKL hydrophobic cluster (Figure 1, top panel). of LEPR’s IGD (amino acids 325C328) drastically reduced its biological but not binding activity, indicating the importance of this Neridronate region for connection with leptin’s site III. FRET (fluorescence resonance energy transfer) microscopy experiments have documented the transient FRET signalling happening upon exposure to leptin results not from binding of the ligand, but from ligand-induced oligomerization of LEPRs mediated by leptin’s site III. polymerase. The mutated create was then digested with DpnI restriction enzyme, which is specific to methylated and hemi-methylated DNA (target sequence: 5-Gm6ATC-3), in order to break down the template and to select for mutations comprising synthesized DNA. The plasmids were then transfected into XL1-proficient cells. Five colonies of each mutant were screened for mutation, using the specific restriction site designed, and exposed at least 80% effectiveness. Two colonies of each mutant were sequenced and confirmed to contain the mutation but no undesirable misincorporation of nucleotides. XL1-proficient cells were transformed with the mutated plasmids and LEP cultivated in 5C10?ml of LuriaCBertani (broth) medium and plasmids were isolated. Mon105-proficient cells were then transformed with the mutated plasmids and utilized for manifestation. Site-directed mutagenesis of the IGD m (mouse) LEPRb cDNA (kindly provided by Dr C. Bj?rbaek, Harvard Medical School, Boston, MA, U.S.A.) was subcloned into the mammalian manifestation vector pEYFP-N1 (where EYFP stands for enhanced yellow fluorescent protein) (ClonTech Ozyme, Saint Quentin en Yvelines, France). Mutagenesis of the different residues in the IGD was performed using the Stratagene QuickChange kit, as already described. Designed primers are demonstrated in Supplementary Table S1 at http://www.BiochemJ.org/bj/391/bj3910221add.htm. Amino acids VFTT (325C328) of the LEPR were Neridronate changed to encode the aliphatic amino acid alanine. The mutagenesis process included 18 PCR cycles using polymerase. Mutated sequences were confirmed by DNA sequencing. Expression, refolding and purification of human and ovine leptins and their mutants The recombinant WT or mutated human leptins with an extra methionineCalanine (methionine is usually cleaved by the bacteria) at the N-terminus were expressed in a 2.5?litre culture. IBs (inclusion bodies) were then prepared as explained previously [15,16] and frozen. Subsequently, IBs obtained from 2.5?litres of bacterial culture were solubilized in 300?ml of 4.5?M urea and 40?mM Tris base containing 10?mM cysteine. In the case of ovine leptin or its mutants, Neridronate the IBs obtained from 1.0?litre of bacterial culture were solubilized in a similar manner in 200?ml. The pH of the solution was adjusted to 11.3 with NaOH. After 2?h of stirring at 4?C, three volumes of 0.67?M arginine were added to a final concentration of 0.5?M and stirred for an additional 1.5?h. Then, the solution was dialysed against 10?litres of 10?mM Tris/HCl, pH?9 (human leptin muteins), or 10?mM Tris/HCl, pH?8 (ovine leptin muteins), for 60?h, with five or six external solution exchanges. The protein was then applied at maximal circulation rate (400C500?ml/h) on to a Q-Sepharose column (30?ml bead volume) and pre-equilibrated with the respective buffer. The breakthrough fraction, which contained no Neridronate leptin, was discarded, and the assimilated protein was eluted in a stepwise manner (50, 100, 150 and 400?mM NaCl in 10?mM Tris/HCl, pH?9 or 8). Fractions (50?ml) were collected and protein concentration was determined by absorbance at 280?nm. Determination of purity and monomer content SDS/PAGE was Neridronate performed as explained by Laemmli  in a 15% (w/v) polyacrylamide gel under reducing conditions. The gel was stained with Coomassie Amazing Blue R. Gel filtration chromatography was performed on a Superdex? 75 HR 10/30 column with 0.2?ml aliquots of the Q-Sepharose-column-eluted fraction using TN buffer (25?mM Tris/HCl and 150?mM NaCl, pH?8). Reverse-phase chromatography was performed using a Vydax column developed with a gradient of 0.1% (v/v) TFA (trifluoroacetic acid) in water (solvent A) and 0.1% TFA in methyl cyanide (solvent B). Determination of CD spectra The CD spectra were measured with an AVIV model 62A DS CD spectrometer (Aviv Associates, Lakewood, NJ, U.S.A.) using a 0.020?cm rectangular QS Hellma cuvette as described previously . Numerous binding assays Determination of the leptinCchLBD complex stoichiometry by gel filtration chromatography, kinetic measurements of chLBDCleptin interactions using SPR (surface plasmon resonance) methodology .