<p>(<b>A</b>) Inclusion bodies from 2.5 L fermentation culture were prepared and resuspended in 100 ml of DDW. Aliquots (corresponding to 0.8, 1.6, 3.2, 4.0, 8.0 and 12.0 µl per lane, from left to right) were separated by 15% SDS-PAGE in presence of β-mercaptoethanol. The molecular mass markers from the bottom up (last lane on the right) are (in kDa): 10, 15, 20, 25, 37, 50, 75, 100, 150 and 250. (<b>B</b>) SDS-PAGE (15%) of purified SRLA run after lyophilization in the absence (lanes 1–2 from the left) or presence (lanes 4–5 from the left) of β-mercaptoethanol (ME) at 2 concentrations: lanes 1 and 4–5 µg, lanes 2 and 5–10 µg. Lane 3– molecular weight markers (see above). (<b>C</b>) SDS-PAGE (10%) of purified PEG-SRLA run after lyophilization in the absence (lanes 1–2 from the left) or presence (lanes 4–5 from the left) of β-mercaptoethanol (ME) at 2 concentrations: lanes 1 and 4–5 µg, lanes 2 and 5–10 µg. Lane 3– molecular weight markers (see above); (<b>D</b>) Gel-filtration analysis of the purified SRLA on analytical Superdex 75 column pre-equilibrated with TN buffer, pH 8. The main peak with retention time of 15.93 min corresponds to monomer and the preceding small shoulder to dimer. (<b>E</b>) Gel-filtration analysis of the purified PEG-SRLA on analytical Superdex 200 column pre-equilibrated with TN buffer, pH 8. The main peak with retention time of 14.93 min corresponds to mono-pegylated PEG-SRLA and the preceding small shoulder to double-pegylated PEG-SRLA. To estimate the molecular mass shown in (D) and (E) the columns were calibrated with BSA (66 kDa), rat CNTF (22 kDa) and human leptin (16 kD); (<b>F</b>) Competitive non-radioactive receptor-binding assay of SRLA and PEG-SRLA. Binding of biotinylated human leptin to immobilized human leptin binding domain (hLBD) consisting of the amino acids 428–635 of human leptin receptor <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086744#pone.0086744-Sandowski1" target="_blank">[51]</a> was performed in the presence of the indicated protein SRLA or PEG-SRLA concentrations. The experiment was carried out in triplicates and the results are presented as mean ± SEM. As the variations in this assay was very small the error bars are not seen; (<b>G</b>) Biological activity of SRLA and PEG-SRLA. The experiment was performed in BAF/3 cells stably transfected with the chimeric leptin receptor construct consisting of the extra-cellular and transmembrane domain of the murine leptin receptor with the intracellular domain of the human βc receptor. Synchronized cells were grown for 48 h in the presence of rat leptin (50 pg/well) and various concentrations of SRLA or PEG-SRLA. The number of cells was then determined by the MTT method. In both bioassays the experiment was carried out in triplicates and the results are presented as mean ± SEM. Detailed description of the binding experiments and the bioassay is provided in our former paper <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086744#pone.0086744-Shpilman1" target="_blank">[28]</a>.</p
