Chromatographic methods for the isolation of, and refolding of proteins from, Escherichia coli inclusion bodies.
New methods for the chromatographic isolation of inclusion bodies directly from crude Escherichia coli homogenates and for the refolding of denatured protein are presented. The traditional method of differential centrifugation for the isolation of purified inclusion bodies is replaced by a single gel-filtration step. The principle is that the exclusion limit of the gel particles is chosen such that only the inclusion bodies are excluded, i.e., all other components of the crude homogenate penetrate the gel under the conditions selected. In the novel column refolding process, a decreasing gradient of denaturant (urea or Gu-HCl), combined with an increasing pH gradient, is introduced into a gel-filtration column packed with a gel medium that has an exclusion limit lower than the molecular mass of the protein to be refolded. A limited sample volume of the protein, dissolved in the highest denaturant concentration at the lowest pH of the selected gradient combination, is applied to the column. During the course of elution, the zone of denatured protein moves down the column at a speed approximately threefold higher than that of the denaturant. This means that the protein sample will gradually pass through areas of increasingly lower denaturant concentrations and higher pH, which promotes refolding into the native conformation. The shape and slope of the gradients, as well as the flow rate, will influence the refolding rate and can be adjusted for different protein samples. The principle is illustrated using a denatured recombinant scFv fusion protein obtained from E. coli inclusion bodies.
Gu
Zhenyu
Z
Institute of Process Engineering, Chinese Academy of Sciences, Beijing, China.
Weidenhaupt
Marianne
M
Ivanova
Natalia
N
Pavlov
Michail
M
Xu
Bingze
B
Su
Zhi-Guo
ZG
Janson
Jan-Christer
JC
D002498:Centrifugation; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D002845:Chromatography; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies / Q000378:metabolism*; D008957:Models, Genetic; D017510:Protein Folding; D014508:Urea / Q000494:pharmacology
D014508:Urea