Overexpression of rnc gene and purification of RNaseIII.
The reason for low content of RNaseIII in E. coli is that RNaseIII has a negative feedback action on its own synthesis by cutting off the transcripts of its own gene, rnc, at 5'-terminal. On this basis, the scheme for overproduction of RNaseIII was designed. The 5'-flanking sequence of rnc gene transcribing to form a secondary structure which could be degraded by RNaseIII was removed, and the whole coding sequence, including the translational initiation signal, was reserved and put under the control of lambda PL promoter. The constructed plasmid pCR21, which contain the recombined rnc gene, overproduced RNaseIII, which covered over 65% of the total cell protein and formed inclusive bodies in E. coli after induction at 42 degrees C. By using the characteristics of solubility of the protein, electrophoretic pure RNaseIII was obtained with a simple procedure, including lysis of the bacterial cells, washing precipitates of the lysate repeatedly at low temperature and low salt concentration, and dissolving and passing through Q-Sepharose FF column in high salt concentration at room temperature. The yield of purified RNaseIII was 10-12 mg per 100 ml culture, and lambda sib transcripts were cut at special sites. RNaseIII possessing the activity of binding ATP is reported.
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Department of Biochemistry, Fourth Military Medical University, Xi'an, Shaanxi, China.
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D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D004722:Endoribonucleases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D029968:Escherichia coli Proteins; D015870:Gene Expression; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics; D043244:Ribonuclease III
D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D000255:Adenosine Triphosphate; D004722:Endoribonucleases; D043244:Ribonuclease III; C468366:ribonuclease III, E coli