Winegar D A, Ohmstede C A, Chu L, Reep B, Lapetina E G
Division of Cell Biology, Burroughs Wellcome Co., Research Triangle Park, North Carolina 27709.
J Biol Chem. 1991 Mar 5;266(7):4375-80.
Polyclonal antisera were generated against synthetic peptides corresponding to distinct regions of the rap 1 protein sequences. A "rap 1-common" antiserum, prepared against an 18-amino acid segment of the rap 1a protein near the proposed GTP-binding region, reacted with both rap 1a and rap 1b recombinant proteins expressed in Escherichia coli and with two low molecular weight GTP-binding proteins of 22 and 24 kDa in unstimulated human platelets. An antiserum raised against a carboxyl-terminal peptide of rap 1b containing the putative site of post-translational processing reacted strongly with bacterial-expressed recombinant rap 1b and with a 24-kDa GTP-binding protein in platelets, but not with recombinant rap 1a or a 22-kDa GTP-binding protein. The mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this rap 1b immunoreactive protein coincided with that of bacterial-expressed rap 1b and not with the faster migrating form of rap 1b that incorporates radioactivity from [3H]mevalonic acid in the insect/baculovirus system. This suggests that our rap 1b-specific antiserum recognizes only one form of rap 1b, that which has not undergone carboxyl-terminal post-translational processing.
针对与rap 1蛋白序列不同区域相对应的合成肽产生了多克隆抗血清。一种“rap 1共同”抗血清是针对rap 1a蛋白靠近拟议的GTP结合区域的一个18个氨基酸片段制备的，它与在大肠杆菌中表达的rap 1a和rap 1b重组蛋白以及未刺激的人血小板中两种分子量分别为22 kDa和24 kDa的低分子量GTP结合蛋白发生反应。一种针对rap 1b羧基末端肽产生的抗血清，该肽含有假定的翻译后加工位点，它与细菌表达的重组rap 1b以及血小板中的一种24 kDa GTP结合蛋白强烈反应，但不与重组rap 1a或一种22 kDa GTP结合蛋白反应。这种rap 1b免疫反应性蛋白在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上的迁移率与细菌表达的rap 1b一致，而与在昆虫/杆状病毒系统中掺入[3H]甲羟戊酸放射性的迁移较快的rap 1b形式不一致。这表明我们的rap 1b特异性抗血清只识别一种rap 1b形式，即未经历羧基末端翻译后加工的形式。
