26S蛋白酶体亚基9的分子克隆与表达

Molecular cloning and expression of subunit 9 of the 26S proteasome.

作者信息

Hoffman L, Rechsteiner M

机构信息

Department of Biochemistry, University of Utah School of Medicine, Salt Lake City 84132, USA.

出版信息

FEBS Lett. 1997 Mar 10;404(2-3):179-84. doi: 10.1016/s0014-5793(97)00126-9.

DOI:10.1016/s0014-5793(97)00126-9

PMID:9119060

Abstract

Seven peptides from subunit 9 (S9) of the human 26S proteasome were sequenced and this information was used to clone a HeLa cDNA that encodes the 46 kDa subunit. Rabbit polyclonal antisera were made against a ubiquitin fusion protein containing 12 amino acids from S9 and against a full-length S9 expressed in E. coli. Western blot analysis showed that the S9-specific antibodies bound the 26S proteasome and its regulatory complex separated on non-denaturing gels. In SDS-PAGE samples of the two complexes, the S9-specific antibodies bound a single 46 kDa subunit. Thus, a cDNA encoding a novel 26S protease subunit has been isolated, sequenced, and expressed.

摘要

对人26S蛋白酶体亚基9（S9）的7个肽段进行了测序，并利用这些信息克隆了一个编码46 kDa亚基的HeLa细胞cDNA。制备了兔多克隆抗血清，分别针对含有S9的12个氨基酸的泛素融合蛋白以及在大肠杆菌中表达的全长S9。蛋白质印迹分析表明，S9特异性抗体与在非变性凝胶上分离的26S蛋白酶体及其调节复合物结合。在这两种复合物的SDS-PAGE样品中，S9特异性抗体与一个单一的46 kDa亚基结合。因此，已分离、测序并表达了一个编码新型26S蛋白酶亚基的cDNA。