Obtaining highly purified intrinsically disordered protein by boiling lysis and single step ion exchange.
Intrinsically disordered proteins (IDPs) is a term used to describe proteins that do not have a well-defined tertiary structure. IDPs have many roles such as in cell cycle control (p53), neuronal signal transmission (myelin basic protein), and protein stability (dehydrins). Producing recombinant IDPs in bacteria for nuclear magnetic resonance (NMR) studies is problematic because the lack of stable tertiary structure makes them excellent substrates for bacterial proteases, which will cause loss in yield. We have developed a two-step method to produce the grape dehydrin K(2) and YSK(2) using Escherichia coli. Dehydrins are expressed by certain plants in response to dehydration, increased salinity, or low temperatures. Purification of 10 mg/L (K(2)) and 15 mg/L (YSK(2)) was performed by boiling bacterial pellets to lyse the cells, remove most of the contaminating proteins, and denature bacterial proteases. This resulted in protein purity comparable to that produced by sonication and nickel affinity chromatography. Boiling was followed by cation exchange chromatography to remove the remaining trace contaminants. The sample was shown to be more than 95% pure by reversed-phase high-performance liquid chromatography. The method presented here can easily be adapted to the purification of other IDPs and heat-stable proteins without requiring multiple chromatography steps or the use of protease inhibitors.
Livernois
Alexandra M
AM
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ont. N1G2W1, Canada.
Hnatchuk
Daniel J
DJ
Findlater
Emma E
EE
Graether
Steffen P
SP
D002852:Chromatography, Ion Exchange / Q000379:methods*; D015870:Gene Expression; D010940:Plant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D044366:Transition Temperature; D027843:Vitis / Q000235:genetics / Q000378:metabolism
D010940:Plant Proteins; D011994:Recombinant Proteins; C070556:dehydrin proteins, plant