Rydlander L, Ziegler E, Bergman T, Schöberl E, Steiner G, Bergman A C, Zetterberg A, Marberger M, Björklund P, Skern T, Einarsson R, Jörnvall H
Division of Research and Development, Beki AB, Bromma, Sweden.
Eur J Biochem. 1996 Oct 15;241(2):309-14. doi: 10.1111/j.1432-1033.1996.00309.x.
Tissue-polypeptide-specific antigen (TPS) from the human colon adenocarcinoma cell line WiDr was purified using the monoclonal antibody M3 as a probe. Upon SDS/polyacrylamide gradient gel electrophoresis, several TPS-positive bands were detected (corresponding to 13 kDa, 22 kDa and a doublet at 42 kDa). The 13-kDa moiety was purified about 30,000-fold by a 5-step protocol. The electro-phoretically homogeneous component was obtained in a 7% yield of the total TPS activity of the crude extract. N-terminal sequence analysis showed the presence of an N-terminally truncated molecule and identified the 13-kDa TPS component as a fragment of human cytokeratin 18, with a major from starting at position 284 of the parent molecule. Laser-desorption mass spectrometry showed the presence of one major component with a molecular mass corresponding to a C-terminal end close to position 396 (which gives 12776 Da for the form with non-truncated N-terminus). The M3 antibody was also used to screen a human prostate cDNA lambda gt11 library. Four identical phage clones were detected, each producing a fusion protein with beta-galactosidase and the M3-positive component. PCR amplification showed the presence of an approximately 1200-bp insert, and sequence analysis revealed it to contain a 996-nucleotide fragment corresponding to residues 103-429 of human cytokeratin 18 (plus a non-coding human desmin artifact fragment). Smaller fragments, engineered by PCR and expressed as fusion proteins using the pET3xc vector in Escherichia coli, showed that the M3 epitope is localized to cytokeratin 18, residues 322-340. Two other TPS-active monoclonal antibodies were localized to cytokeratin 18 with similar techniques, ascribing an epitope (to M21) to residues 414-429 and another (to M24) to residues 139-297. Combined, the results demonstrate that TPS reactivity is derived from specific epitopes of human cytokeratin 18.
使用单克隆抗体M3作为探针，对人结肠腺癌细胞系WiDr中的组织多肽特异性抗原（TPS）进行了纯化。在SDS/聚丙烯酰胺梯度凝胶电泳中，检测到几条TPS阳性条带（对应于13 kDa、22 kDa以及42 kDa处的一条双条带）。通过五步方案将13 kDa部分纯化了约30000倍。以粗提物总TPS活性7%的产率获得了电泳均一的组分。N端序列分析显示存在一个N端截短的分子，并将13 kDa的TPS组分鉴定为人细胞角蛋白18的一个片段，其主要起始于亲本分子的第284位。激光解吸质谱显示存在一个主要组分，其分子量对应于接近第396位的C端（对于N端未截短的形式为12776 Da）。M3抗体还用于筛选人前列腺cDNA λgt11文库。检测到四个相同的噬菌体克隆，每个克隆都产生一种与β-半乳糖苷酶和M3阳性组分的融合蛋白。PCR扩增显示存在一个约1200 bp的插入片段，序列分析表明它包含一个996核苷酸的片段，对应于人细胞角蛋白18的第103 - 429位残基（加上一个非编码的人结蛋白伪片段）。通过PCR构建并使用pET3xc载体在大肠杆菌中表达为融合蛋白的较小片段表明，M3表位定位于细胞角蛋白18的第322 - 340位残基。使用类似技术将另外两种具有TPS活性的单克隆抗体定位于细胞角蛋白18，将一个表位（对于M21）定位于第414 - 429位残基，另一个（对于M24）定位于第139 - 297位残基。综合起来，结果表明TPS反应性源自人细胞角蛋白18的特定表位。
