title: Expression of recombinant MDA-BF-1 with a kinase recognition site and a 7-histidine tag for receptor binding and purification.
abstract: Prostate cancer metastasizes predominantly to bone, where it induces osteoblastic lesions. Paracrine factors secreted by the metastatic cancer cells are thought to mediate these events. We previously isolated a novel bone metastasis-related factor (MDA-BF-1) from bone marrow aspirate samples from patients with prostate cancer and bone metastasis, and found that this factor stimulated osteoblast differentiation, possibly by interacting with a receptor on the osteoblasts. Identifying this putative MDA-BF-1 receptor biochemically requires the expression of MDA-BF-1 for receptor binding assays and for the preparation of a ligand-affinity column. We tagged MDA-BF-1 with a peptide containing a protein kinase A phosphorylation site plus a 7-histidine sequence to facilitate the labeling of MDA-BF-1 for receptor binding assay and the binding of MDA-BF-1 to an immobilized metal affinity column. The recombinant MDA-BF-1 protein (MDA-BF1-kinase-his) was expressed in Sf9 cells using a baculovirus expression system. About 0.8 mg of purified MDA-BF1-kinase-his protein was obtained from 4 x 10(8) Sf9 cells. MDA-BF1-kinase-his can be phosphorylated by PKA with a specific activity around 10(5)cpm/mug protein. Receptor binding assays using this (32)P-labeled MDA-BF-1 showed that MDA-BF-1 bound to membranes prepared from Saos-2, an osteosarcoma cell line, and C2C12, a mouse pluripotent mesenchymal precursor cell line that can be induced to become osteoblast by BMP-2. In contrast, MDA-BF-1 did not bind to membranes from PC-3 human prostate cancer cells or HEK293 human embryonic kidney cells. These observations suggest that the MDA-BF-1 receptor is expressed in cells of osteoblastic lineage. In addition to its use as a ligand for receptor binding assays, a ligand affinity column can be prepared by binding MDA-BF1-kinase-his to an IMAC for the purification of MDA-BF-1 receptor.
authors.affiliation: Department of Molecular Pathology, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA.
authors.forename: Albert K
authors.initials: AK
authors.lastname: Liang
authors.forename: Jonathan
authors.initials: J
authors.lastname: Liu
authors.forename: Stephen A
authors.initials: SA
authors.lastname: Mao
authors.forename: Vince S
authors.initials: VS
authors.lastname: Siu
authors.forename: Yu-Chen
authors.initials: YC
authors.lastname: Lee
authors.forename: Sue-Hwa
authors.initials: SH
authors.lastname: Lin
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics; D066246:ErbB Receptors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D015870:Gene Expression / Q000235:genetics; D006639:Histidine / Q000235:genetics; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D009363:Neoplasm Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011485:Protein Binding; D020893:Receptor, ErbB-3; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: C507128:MDA-BF-1, human; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D011993:Recombinant Fusion Proteins; D006639:Histidine; D066246:ErbB Receptors; D020893:Receptor, ErbB-3; D017868:Cyclic AMP-Dependent Protein Kinases