Kuroki M, Murakami M, Wakisaka M, Ikeda S, Oikawa S, Oshima T, Nakazato H, Kosaki G, Matsuoka Y
First Department of Biochemistry, School of Medicine, Fukuoka University, Japan.
Immunol Invest. 1992 Jun;21(3):241-57. doi: 10.3109/08820139209072262.
Immunoreactivities of recombinant carcinoembryonic antigen (CEA) proteins expressed in Escherichia coli (E. coli) were analyzed in relation to the CEA domain structure [domains N, I (A1-B1), II (A2-B2), III (A3-B3) and M]. We reconstructed in a prokaryotic expression vector, pUCPL-cI, the cDNAs fro CEA-N, CEA-I, CEA-II, and CEA-III-M. The latter three were expressed as fusion products with bacterial beta-galactosidase. The recombinant proteins were solubilized by sonication in 1% sodium dodecyl sulfate (SDS) and purified by preparative SDS-polyacrylamide gel electrophoresis followed by electroelution. Their molecular weights judged from Western blotting coincided with those calculated from their cDNA sequences, respectively. By solid-phase enzyme immunoassays, the immunoreactivities of the purified recombinant proteins were tested with 21 distinct anti-CEA monoclonal antibodies (MAbs) which had been found to recognize the peptide epitopes of the CEA molecule and to be reactive with the recombinant CEA proteins expressed in Chinese hamster ovary (CHO) cells. Fourteen of the 21 MAbs reacted with the recombinant CEA proteins expressed in E. coli and confirmed the localization of the epitopes identified by using the recombinant CEA proteins expressed in CHO cells. The reactivities of 5 MAbs with the recombinant proteins expressed in E. coli were remarkably low when compared with those of the proteins expressed in CHO cells but also confirmed the localization of the epitopes identified with the recombinant CEA proteins expressed in CHO cells. The remaining 2 MAbs did not react with any recombinant protein expressed in E. coli. These results indicate that the fusion CEA-proteins expressed in E. coli are useful in the localization of the epitopes on the polypeptide chains when they reacted with the MAbs tested. However, one third of the epitopes of CEA peptides may be profoundly affected by the presence of disulfide bonds and/or sugar chains which do not seem to be formed well in E. coli.
分析了在大肠杆菌（E. coli）中表达的重组癌胚抗原（CEA）蛋白的免疫反应性与CEA结构域[结构域N、I（A1 - B1）、II（A2 - B2）、III（A3 - B3）和M]的关系。我们在原核表达载体pUCPL - cI中构建了CEA - N、CEA - I、CEA - II和CEA - III - M的cDNA。后三者作为与细菌β - 半乳糖苷酶的融合产物表达。通过超声处理将重组蛋白溶解在1%十二烷基硫酸钠（SDS）中，并通过制备性SDS - 聚丙烯酰胺凝胶电泳随后电洗脱进行纯化。从蛋白质印迹法判断的它们的分子量分别与从其cDNA序列计算出的分子量一致。通过固相酶免疫测定法，用21种不同的抗CEA单克隆抗体（MAbs）测试了纯化的重组蛋白的免疫反应性，这些单克隆抗体已被发现可识别CEA分子的肽表位并与在中国仓鼠卵巢（CHO）细胞中表达的重组CEA蛋白反应。21种MAbs中的14种与在大肠杆菌中表达的重组CEA蛋白反应，并证实了使用在CHO细胞中表达的重组CEA蛋白鉴定的表位的定位。与在CHO细胞中表达的蛋白相比，5种MAbs与在大肠杆菌中表达的重组蛋白的反应性明显较低，但也证实了使用在CHO细胞中表达的重组CEA蛋白鉴定的表位的定位。其余2种MAbs与在大肠杆菌中表达的任何重组蛋白均无反应。这些结果表明，当在大肠杆菌中表达的融合CEA蛋白与所测试的MAbs反应时，它们可用于定位多肽链上的表位。然而，CEA肽的三分之一表位可能会受到二硫键和/或糖链的显著影响，而这些在大肠杆菌中似乎无法很好地形成。
