Grauer L S, Lawler K D, Marignac J L, Kumar A, Goel A S, Wolfert R L
Hybritech Incorporated, Beckman Coulter, Inc., San Diego, California 92196-9006, USA.
Cancer Res. 1998 Nov 1;58(21):4787-9.
An alternatively spliced variant of prostate-specific membrane antigen (PSMA) designated PSM' was originally described following identification of its mRNA in normal prostate. We have purified the PSM' protein from LNCaP cells using two immunoaffinity columns in tandem. The first column contained a monoclonal antibody (7E11) that was reactive with the NH2 terminus of PSMA, which specifically depleted the LNCaP lysate of full-length PSMA. The nonbinding fraction was then passed over a second column composed of a monoclonal antibody (PEQ226.5), the epitope of which was located within the 134-437 domain of PSMA and shared with PSM'. The protein eluted from the second immunoaffinity column produced a Mr 95,000 band on SDS-PAGE, which was slightly lower than the full-length PSMA at Mr 100,000. The band was NH2-terminally sequenced through 15 residues, and the assigned sequence coincided with the predicted sequence for PSM' protein minus the first two NH2 terminus amino acids. The PSM' protein, therefore, began with residue 60 of PSMA (alanine). LNCaP cells were fractionated, and PSM' was localized to the cytoplasm.
前列腺特异性膜抗原（PSMA）的一种可变剪接变体，命名为PSM'，最初是在正常前列腺组织中发现其mRNA后被描述的。我们利用两个串联的免疫亲和柱从LNCaP细胞中纯化了PSM'蛋白。第一个柱子含有一种与PSMA的NH2末端反应的单克隆抗体（7E11），它特异性地去除了LNCaP裂解物中的全长PSMA。然后将未结合部分通过由单克隆抗体（PEQ226.5）组成的第二个柱子，该抗体的表位位于PSMA的134 - 437结构域内且与PSM'共享。从第二个免疫亲和柱洗脱的蛋白在SDS - PAGE上产生了一条分子量为95,000的条带，略低于全长PSMA的分子量100,000。对该条带进行了15个残基的NH2末端测序，所得序列与PSM'蛋白预测序列相符，但缺少前两个NH2末端氨基酸。因此，PSM'蛋白从PSMA的第60个残基（丙氨酸）开始。对LNCaP细胞进行了分级分离，并将PSM'定位到细胞质中。
