Acta Academiae Medicinae Sinica

Acta Academiae Medicinae Sinica

Acta Academiae Medicinae Sinica ›› 2011, Vol. 33 ›› Issue (6): 638-643.doi: 10.3881/j.issn.1000-503X.2011.06.011

• Original Articles • Previous Articles     Next Articles

Prokayotic Expression and Purification of Mitochondrial Transcription Complex Proteins

LIU Guang, YANG Rui-feng, SHI Bing-yang, LIU De-pei   

  1. National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences,CAMS and PUMC, Beijing 100005, China
  • Received:2011-09-05 Revised:2012-01-04 Online:2011-12-20 Published:2011-12-20
  • Contact: LIU De-pei
  • Supported by:

    Supported by the National Basic Research Program of China (973 Program) (2011CB503902) and the National Laboratory of Medical Molecular Biology (2060204)

Abstract: Objective To obtain human mitochondrial transcription factor A (TFAM), mitochondrial transcription factor B1 (TFB1M), and mitochondrial transcription factor B2 (TFB2M) that were expressed efficiently in E.coli BE21 and to purify the target proteins. Methods TFAM, TFB1M, and TFB2M segments were designed and synthesized. After having been sequenced, the reconstructed expression vectors were constructed by enzyme digestion and by cloning into an expression vector pET42a. Then the reconstructed vectors were transformed into E.coli BL21. Recombinant glutathione S transferase (GST) fusion proteins were expressed via the induction of IsoPropyl β-D-ThioGalactoside (IPTG) and purified by glutathione Sepharose 4B. Results The expression plasmids of pET42a-TFAM, pET42a-TFB1M, and pET42a-TFB1M were successfully constructed. The sequences of the cloned gene segments were identical with GenBank reported.The protein bands with relative molecular masses of 56.000, 67.000, and 69.000 appeared on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) after the expressed GST-TFAM, GST-TFB1M, and GST-TFB2M fusion proteins were separated by SDS-PAGE.The expressed fusion proteins were purified to high purity. Conclusion The recombinant plasmids pET42a-TFAM,pET42a-TFB1M, and pET42a-TFB2M were successfully costructed, and the GST-fused target proteins were prepared.

Key words: mitochondrial transcription factor A, mitochondrial transcription factor B1, mitochondrial transcription factor B2, fusion protein, purification

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