Zhao Yan, Duan Su-su, Che Chang-yan, Zhang Lei, Zhang Yun, Wang Shu-hui, Liu Li
Department of Microbiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Science, Beijing 100005, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2008 Jan;24(1):27-9.
Cytokine receptor-like factor 3 (CRLF3) is a novel gene cloned from K562 cell line. The aim of the current study is to observe CRLF3 expression and subcellular localization in 293T cells and to express and purify the CRLF3 fusion protein in E.coli.
The plasmid pCMV-myc-CRLF3 was transiently transfected into 293T cell. The expression and localization of CRLF3 protein was observed by immunostaining approach. CRLF3 was also cloned into pGEX-4T-1 vector. The GST-CRLF3 fusion protein was expressed in E.coli and purified by GST affinity chromatography.
CRLF3 protein was highly expressed in transfected 293T cells. CRLF3 protein was distributed in both cytoplasm and cell membrane. The GST-CRLF3 fusion protein was expressed as a M(r) 74 000 protein and then successfully purified from E.coli cells.
Overexpressed CRLF3 is a cytoplasmic protein that distributes in both cytoplasm and cell membrane in mammalian cells. The recombinant GST-CRLF3 fusion protein had been isolated and could be used for further antibody production and functional characterization.
细胞因子受体样因子3（CRLF3）是从K562细胞系克隆的一个新基因。本研究的目的是观察CRLF3在293T细胞中的表达及亚细胞定位，并在大肠杆菌中表达和纯化CRLF3融合蛋白。
将质粒pCMV-myc-CRLF3瞬时转染至293T细胞。采用免疫染色法观察CRLF3蛋白的表达及定位。将CRLF3也克隆至pGEX-4T-1载体。GST-CRLF3融合蛋白在大肠杆菌中表达，并通过GST亲和层析进行纯化。
CRLF3蛋白在转染的293T细胞中高表达。CRLF3蛋白分布于细胞质和细胞膜。GST-CRLF3融合蛋白表达为分子量74 000的蛋白，随后成功从大肠杆菌细胞中纯化出来。
过表达的CRLF3是一种细胞质蛋白，在哺乳动物细胞的细胞质和细胞膜中均有分布。重组GST-CRLF3融合蛋白已被分离，可用于进一步制备抗体和功能鉴定。
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