Osmo-expression and fast two-step purification of Escherichia coli translation termination factor RF-3.
The gene for the translation termination factor RF-3 in Escherichia coli has recently been cloned and sequenced. Only small amounts of the protein have been purified until now, not sufficient for detailed investigation of the structure and function of this factor. For such studies, we have developed an overexpression system and a purification procedure suitable for large quantities of RF-3. The gene prfC was cloned into the osmo-inducible plasmid pOSEX3 and subsequently transformed into the E. coli strain MKH13. The expression of prfC in this plasmid, which is under the control of the osmotic pressure in the growth medium, leads to a level of RF-3 more than 100-times higher than that in wild-type cells. Using a new two-step FPLC protein purification procedure consisting of ion-exchange chromatography on Q-Sepharose FF and S-Sepharose HP, we obtain 220 mg pure RF-3 from 10 g overproducing cells, corresponding to 55 mg RF-3/l medium. The identity of the purified protein was confirmed by matrix-assisted laser desorption/ionisation mass spectrometry of tryptolytic fragments and by N-terminal amino acid sequencing. The activity of the purified factor was tested in vitro by measuring the stimulation of RF-2 dependent formylmethionine release from a ribosomal termination complex and the binding capacity of GTP and GDP. All assays showed that the purified RF-3 was highly active with a specific activity of approximately 2000 units/mg.
Mortensen
K K
KK
Department of Chemistry, Aarhus University, Denmark.
Hansen
H F
HF
Grentzmann
G
G
Buckingham
R H
RH
Sperling-Petersen
H U
HU
D000595:Amino Acid Sequence; D002847:Chromatography, Agarose; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors / Q000235:genetics; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D008969:Molecular Sequence Data; D009239:N-Formylmethionine / Q000378:metabolism; D009997:Osmotic Pressure; D010454:Peptide Termination Factors / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000378:metabolism
D029968:Escherichia coli Proteins; D010454:Peptide Termination Factors; D011994:Recombinant Proteins; C087971:peptide-chain-release factor 3; C489007:prfC protein, E coli; D006153:Guanosine Diphosphate; D009239:N-Formylmethionine; D006160:Guanosine Triphosphate; D014357:Trypsin