[Isolation of Tara protein and its gene cloning].
To isolate and identify TRF1 immunoprecipitating protein complex and to clone the candidate gene.



The co-immunoprecipitation assay was employed to isolate TRF1 protein complex and the immunoprecipitate was subjected to MALDI-TOF mass spectrometry for protein identification. The candidate gene was amplified by temperature-gradient PCR from human testis cDNA library and then cloned into pEGFP-C2 vector for eukaryotic expression. The amplified gene was verified by direct sequencing and GFP-tagged protein was confirmed by immunoblotting.



Tara protein with the size of 68 kD was identified from the TRF1 precipitate. The candidate gene amplified from cDNA library was about 1.7 kb as expected. Sequencing demonstrated the amplified fragment had 99.9% of homogenesis with Tara CDS sequence (gi:30474869). GFP-tagged fusion protein was about 100 kD. Tara was diffusely distributed in cytoplasm at interphase and in whole cells at mitotic phase.



Tara might be an interacting protein with TRF1. However, further investigation would be required to confirm if they were bona fide partners.
Lan
Jian-ping
JP
The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China.
Luo
Yi
Y
Zhu
Yuan-yuan
YY
Sun
Jie
J
Lai
Xiao-yu
XY
Li
Jing-yuan
JY
Yu
Jian
J
Shi
Ji-min
JM
Lin
Mao-fang
MF
Huang
He
H
D003001:Cloning, Molecular; D006367:HeLa Cells; D006801:Humans; D008840:Microfilament Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D011485:Protein Binding; D035321:Telomeric Repeat Binding Protein 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D008840:Microfilament Proteins; C425471:TRIOBP protein, human; D035321:Telomeric Repeat Binding Protein 1