用于在哺乳动物细胞中表达和分泌FLAG表位标签蛋白的载体。

Vectors for expression and secretion of FLAG epitope-tagged proteins in mammalian cells.

作者信息

Chubet R G, Brizzard B L

机构信息

Scientific Imaging Systems, Eastman Kodak Company, New Haven, CT, USA.

出版信息

Biotechniques. 1996 Jan;20(1):136-41. doi: 10.2144/96201pf01.

DOI:10.2144/96201pf01

PMID:8770418

Abstract

The FLAG peptide, AspTyrLysAspAspAspAspLys, has been used as an epitope tag in a variety of cell types. The modification of the cytomegalovirus (CMV) promoter containing vector, pCMV5, to create two transient expression vectors designed for secretion and intracellular expression of FLAG-fusion proteins in mammalian cells is described. As a functional test, the bacterial alkaline phosphatase gene was cloned into both vectors, and anti-FLAG monoclonal antibodies were used for detection of FLAG epitope-tagged bacterial alkaline phosphatase in mammalian cells. In addition, secreted bacterial alkaline phosphatase was purified from the extracellular medium by anti-FLAG affinity chromatography.

摘要

FLAG肽，即天冬氨酸-酪氨酸-赖氨酸-天冬氨酸-天冬氨酸-天冬氨酸-天冬氨酸-赖氨酸，已在多种细胞类型中用作表位标签。本文描述了对含巨细胞病毒（CMV）启动子的载体pCMV5进行改造，以构建两个用于在哺乳动物细胞中分泌和细胞内表达FLAG融合蛋白的瞬时表达载体。作为功能测试，将细菌碱性磷酸酶基因克隆到这两个载体中，并使用抗FLAG单克隆抗体检测哺乳动物细胞中带有FLAG表位标签的细菌碱性磷酸酶。此外，通过抗FLAG亲和层析从细胞外培养基中纯化分泌的细菌碱性磷酸酶。