Liang Albert K, Liu Jonathan, Mao Stephen A, Siu Vince S, Lee Yu-Chen, Lin Sue-Hwa
Department of Molecular Pathology, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA.
Protein Expr Purif. 2005 Nov;44(1):58-64. doi: 10.1016/j.pep.2005.03.025.
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.
前列腺癌主要转移至骨骼，在骨骼中形成成骨病变。转移性癌细胞分泌的旁分泌因子被认为介导了这些过程。我们之前从患有前列腺癌并发生骨转移的患者的骨髓穿刺样本中分离出一种新型骨转移相关因子（MDA - BF - 1），并发现该因子可能通过与成骨细胞上的受体相互作用来刺激成骨细胞分化。要通过生化方法鉴定这种假定的MDA - BF - 1受体，需要表达MDA - BF - 1用于受体结合测定以及制备配体亲和柱。我们用含有蛋白激酶A磷酸化位点加7个组氨酸序列的肽标记MDA - BF - 1，以利于为受体结合测定标记MDA - BF - 1以及使MDA - BF - 1与固定化金属亲和柱结合。重组MDA - BF - 1蛋白（MDA - BF1 - kinase - his）使用杆状病毒表达系统在Sf9细胞中表达。从4×10⁸个Sf9细胞中获得了约0.8 mg纯化的MDA - BF1 - kinase - his蛋白。MDA - BF1 - kinase - his可被蛋白激酶A磷酸化，比活性约为10⁵ cpm/μg蛋白。使用这种³²P标记的MDA - BF - 1进行的受体结合测定表明，MDA - BF - 1与骨肉瘤细胞系Saos - 2以及可被骨形态发生蛋白-2诱导成为成骨细胞的小鼠多能间充质前体细胞系C2C12制备的膜结合。相比之下，MDA - BF - 1不与PC - 3人前列腺癌细胞或HEK293人胚肾细胞的膜结合。这些观察结果表明MDA - BF - 1受体在成骨细胞系细胞中表达。除了用作受体结合测定的配体之外，还可以通过将MDA - BF1 - kinase - his与固定化金属离子亲和色谱（IMAC）结合来制备配体亲和柱，用于纯化MDA - BF - 1受体。
