[Cloning, expression and purification of His-tagged mouse mitochondrial transcription factor A and preparation of its polyclonal antibody]
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Published on: September 30, 2010
Zhi-feng Liu1, Zi-yun Shao, Jia Xu
1Key Laboratory of Functional Proteomics of Guangdong Province, Southern Medical University, Guangzhou 510515, China. lizf@fimmu.com
Objective:
To express and purify the fusion protein of His-tagged mouse mitochondrial transcription factor A (mtTFA) in prokaryocytes and prepare rabbit anti-mtTFA polyclonal antibody.
Methods:
The total RNA was extracted from mouse liver cells, and the coding sequence of mtTFA without signal peptides was amplified with reverse transcriptional (RT)-PCR. The PCR product was then cloned into the prokaryotic expression vector pET14b. After enzyme digestion and DNA sequencing, the plasmid was transformed into BL21(DE3) competent cells, and IPTG was used to induce the expression of His-tagged mtTFA. The expressed fusion protein was purified with Ni(2+)-NTA His-bind resin and quantified with Bradford method.
Results:
The amplified product was confirmed as the coding sequence of mtTFA without signal peptides by DNA sequencing, and the recombinant expression vector could express His-tagged mtTFA in E.coil following the induction by IPTG. The fusion protein of high purity and rabbit anti-mtTFA serum with high specificity were obtained.
Conclusions:
The prokaryotic expression vector for mtTFA is successfully constructed and His-tagged mtTFA protein and specific polyclonal antibody are obtained, which can be helpful for further investigation of the biological function and signaling pathways involved in the regulation of functional cooperation between the nucleus and mitochondria.
