A novel strategy for the purification of recombinantly expressed unstructured protein domains.
We recently found that the larger parts of the endocytic proteins epsin 1 and AP180 consist of an unstructured polypeptide chain. As a result these segments are completely heat-stable without loss of their functional properties. We have taken advantage of this fact and developed a combined heat lysis and pre-purification procedure after expressing the disordered domains in E. coli. This results in the irreversible denaturation and precipitation of the majority of bacterial proteins. The bacteria are resuspended in a non-denaturing buffer, heated in a boiling water bath and shock-cooled. We demonstrate that this procedure compared to conventional lysis improves both yield and quality of the purified protein.
Kalthoff
Christoph
C
Department of Cell Biology, Center of Anatomy, Hannover Medical School, D-30625, Hannover, Germany. christoph.kalthoff@zellbiologie.mh-han-nover.de
D033942:Adaptor Proteins, Vesicular Transport; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis; D034041:Monomeric Clathrin Assembly Proteins / Q000737:chemistry / Q000302:isolation & purification*; D009479:Neuropeptides / Q000737:chemistry / Q000302:isolation & purification*; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D033921:Vesicular Transport Proteins
D033942:Adaptor Proteins, Vesicular Transport; D002352:Carrier Proteins; D034041:Monomeric Clathrin Assembly Proteins; D009479:Neuropeptides; D011994:Recombinant Proteins; D033921:Vesicular Transport Proteins; C439023:clathrin assembly protein AP180; C114154:epsin