Expression, purification, and characterization of Tara, a novel telomere repeat-binding factor 1 (TRF1)-binding protein.
Tara was originally identified as a binding protein of guanine nucleotide exchange factor Trio. Although Tara may be involved in many fundamental cellular processes, ranging from actin remodeling, directed cell movement, to cell cycle regulation, aging, and cancer, the exact molecular mechanisms are poorly understood. We expressed recombinant Tara in Escherichia coli and purified the protein to approximately 99% purity using affinity chromatography and gel-filtration chromatography. The identity of the purified protein was confirmed by mass spectrometry. Non-denaturing polyacrylamide gel electrophoresis and gel-filtration chromatography showed that Tara forms multimer in vitro. The purified Tara was used to generate polyclonal antibody, which could specifically recognize both the recombinant and endogenous Tara. Using the pull-down assay, we showed that the purified Tara interacted with TRF1, suggesting that the purified protein is functional and biologically active. The availability of purified Tara and anti-Tara antibody provides critical reagents for elucidating Tara's cellular function and its molecular mechanism.
Li
Xiaoxiao
X
The First Affiliated Hospital of Zhejiang University Medical School, 79 Qingchun Road, Hangzhou 310003, China.
Lan
Jianping
J
Zhu
Yuanyuan
Y
Yu
Jian
J
Dou
Zhen
Z
Huang
He
H
D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D013058:Mass Spectrometry; D008840:Microfilament Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D035321:Telomeric Repeat Binding Protein 1 / Q000737:chemistry / Q000378:metabolism
D000906:Antibodies; D008840:Microfilament Proteins; D011994:Recombinant Proteins; C425471:TRIOBP protein, human; D035321:Telomeric Repeat Binding Protein 1