Expression of recombinant MDA-BF-1 with a kinase recognition site and a 7-histidine tag for receptor binding and purification.
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.
Liang
Albert K
AK
Department of Molecular Pathology, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA.
Liu
Jonathan
J
Mao
Stephen A
SA
Siu
Vince S
VS
Lee
Yu-Chen
YC
Lin
Sue-Hwa
SH
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
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