Cloning and expression of apolipoprotein E3 and its variant apoE2 and apoE4.
In order to obtain three isoforms of apolipoprotein E (apoE), the cDNA encoding apoE3 was obtained by RT-PCR from normal human liver tissue. Site-directed mutagenesis was used to obtain the cDNAs encoding apoE2 and apoE4 isoforms. The 3 cDNAs were subcloned into vector pGEM-3Z and verified by DNA sequencing. The expression recombinant which can express the target protein as a (His) 6-tagged fusion was constructed by subcloning apoE cDNA into vector pT7-PL. The purified proteins were gained by Ni-NTA column. The SDS-PAGE results revealed the 6 His fusion proteins (apoE2, apoE3 and apoE4) were correctly expressed and purified successfully.
Zong
Yiqiang
Y
Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Liu
Zhiguo
Z
Bi
Hao
H
Yao
Yanyi
Y
Guo
Jianli
J
Qu
Shen
S
D053329:Apolipoprotein E2; D053318:Apolipoprotein E3; D053327:Apolipoprotein E4; D001057:Apolipoproteins E / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D003001:Cloning, Molecular; D006801:Humans; D008099:Liver / Q000737:chemistry*; D008969:Molecular Sequence Data; D017354:Point Mutation
D053329:Apolipoprotein E2; D053318:Apolipoprotein E3; D053327:Apolipoprotein E4; D001057:Apolipoproteins E