Prokaryotic expression and purification of mitochondrial transcription complex proteins / 中国医学科学院学报

ABSTRACT

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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 beta-D-ThioGalactoside (IPTG) and purified by glutathione Sepharose 4B.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>The recombinant plasmids pET42a-TFAM, pET42a-TFB1M, and pET42a-TFB2M were successfully constructed, and the GST-fused target proteins were prepared.</p>