Escherichia coli bl21

rBmpA was produced in Escherichia coli BL21 (GE Healthcare, Chicago, IL, USA) using the bacterial expression vector pGEX-6P1 (GE Healthcare) and the following primers with EcoRI and XhoI restriction sites: Forward, 5′-ACGAATTCATGAATAAAATATTGTTGTTGA-3′ and reverse, 5′-AGCTCGTAAATAAATTCTTTAAGAAA-3′ (4 (link),5 (link)). Pure ISOF was provided by Dr Weimin Yang [School of Pharmaceutical Science and Yunnan Key Laboratory of Pharmacology for Natural Products, Kunming Medical University (KMU), Kunming, China] and reconstituted at 1×10⁵ µM/ml in dimethyl sulfoxide (DMSO) for stock solution. LPS and phorbol-12-myristate-13-acetate (PMA) were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany) and ionomycin was purchased from Apollo Scientific Ltd. (Stockport, UK). Recombinant human (rh)IL-4, rhTNF-α and rh-granulocyte macrophage colony-stimulating factor (rhGM-CSF) were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

In vitro binding experiments were performed as described previously (Ebnet et al., 2000 (link)). Briefly, GST-fusion proteins were expressed in Escherichia coli BL21 (GE Healthcare). Bacteria were lysed by passaging through a French pressure cell, and GST-fusion proteins were purified by affinity chromatography. Protein solutions were adjusted to 50% (wt/vol) glycerol and stored at −20°C. For GST pull-down experiments, the prey proteins were generated in vitro using the TNT T7-coupled reticulocyte lysate system (Promega, Madison, WI) in the presence of ³⁵[S]methionine as described by the manufacturer. We incubated 10 μl of the translation reactions with 3 mg of GST-fusion proteins immobilized on glutathione-Sepharose 4B beads (Life Technologies) for 2 h at 4°C under constant agitation. Beads were washed five times with buffer B (10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES], pH 7.2, 100 mM KCl, 1 mM MgCl₂, 0.1% Triton X-100). Bound proteins were eluted by boiling for 5 min in SDS sample buffer, subjected to SDS–PAGE, and analyzed by fluorography.