title: [Prokaryotic expression and purification of mitochondrial transcription complex proteins].
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 beta-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.
  
  
  CONCLUSIONS
  The recombinant plasmids pET42a-TFAM, pET42a-TFB1M, and pET42a-TFB2M were successfully constructed, and the GST-fused target proteins were prepared.
authors.affiliation: National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, CAMS and PUMC, Beijing 100005, China.
authors.forename: Guang
authors.initials: G
authors.lastname: Liu
authors.forename: Rui-Feng
authors.initials: RF
authors.lastname: Yang
authors.forename: Bing-Yang
authors.initials: BY
authors.lastname: Shi
authors.forename: De-Pei
authors.initials: DP
authors.lastname: Liu
mesh_terms: D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D008780:Methyltransferases / Q000235:genetics*; D024101:Mitochondrial Proteins / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics*
chemical_list: D004268:DNA-Binding Proteins; D024101:Mitochondrial Proteins; D011993:Recombinant Fusion Proteins; C499632:TFB1M protein, human; D014157:Transcription Factors; C422562:mitochondrial transcription factor A; D008780:Methyltransferases; C462443:TFB2M protein, human