Refolding proteins by gel filtration chromatography.
We have developed a facile means for the refolding of milligram quantities of purified proteins that employs gel filtration chromatography. We demonstrate by electrophoretic mobility shift and NMR spectroscopy that human ETS-1 protein, bovine ribonuclease A and E. coli integration host factor can be refolded into the native conformation using this technique. We have extended this strategy to the preparation of milligram quantities of macromolecular complexes suitable for structural analysis by NMR spectroscopy or X-ray crystallography. The diverse challenges to overcome in refolding these proteins illustrates the potential of this technique as a general approach for recovery of recombinant proteins produced as insoluble inclusion bodies.
Werner
M H
MH
Laboratory of Chemical Physics, National Institute of Diabetes, Digestive Kidney Diseases, National Institutes of Health, Bethesda, MD 20892.
Clore
G M
GM
Gronenborn
A M
AM
Kondoh
A
A
Fisher
R J
RJ
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D002850:Chromatography, Gel / Q000379:methods*; D004268:DNA-Binding Proteins / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D034982:Integration Host Factors; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D011487:Protein Conformation; D017510:Protein Folding; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000378:metabolism; D050783:Proto-Oncogene Proteins c-ets; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C494041:ETS1 protein, human; D034982:Integration Host Factors; D015345:Oligonucleotide Probes; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D014157:Transcription Factors; D012259:Ribonuclease, Pancreatic