Construction of eukaryotic expression vector of SPAG4L tagged with Myc and His.
- Author:
Yanliang CHEN
;
Zhi ZHENG
;
Jianlong WANG
;
Xiaozhe ZHOU
;
Yan LI
;
Meng YANG
;
Lihua HUANG
;
Xiaowei XING
- Publication Type:Journal Article
- MeSH:
Carrier Proteins;
biosynthesis;
genetics;
Cloning, Molecular;
Female;
Genetic Vectors;
genetics;
HeLa Cells;
Histidine;
genetics;
Humans;
Male;
Proto-Oncogene Proteins c-myc;
genetics;
Recombinant Fusion Proteins;
biosynthesis;
genetics;
isolation & purification;
Transfection
- From:
Chinese Journal of Biotechnology
2013;29(11):1654-1662
- CountryChina
- Language:Chinese
-
Abstract:
The aim of this study is to establish stable transfected cell lines which could produce SPAG4L protein labeled with Myc and His tags in vitro. The open reading frame (ORF) of human SPAG4L was amplified by PCR and the fragments were cloned into eukaryotic expression vector pcDNA3.1/myc-His(-)A. The recombined plasmids of pcDNA3.1/myc-His(-)A/SPAG4L were verified by sequencing and digestion with enzymes. Then, the recombined plasmids were introduced into HeLa cells and screened by G418. Western blotting was performed to detect the expression of SPAG4L and its tags in stable transfected cell lines. SPAG4L and its tags were expressed in the stable cell lines transfected with pcDNA3.1/myc-His(-)A/SPAG4L, but not in the control group. Further study showed that SPAG4L colocalized with the endoplasmic reticulum(ER) marker PDI by immunofluorescence. The stable transfected cell lines established in this study will provide a powerful tool for further studies such as co-immunoprecipitation and pull-down.
