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Schlagwörter:
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Zusammenfassung:
The heterologous expression, isolation and characterization of pore forming protein is one of the most used strategy for understanding permeability properties of the biological membrane into which they are embedded. Some procedures present in literature are so empirical and long to perform, that the user can easily make mistakes. This protocol will teach you how to quantify and therefore optimize the expression of your favorite protein in E. coli using the BL21Gold(de3)ΔABCF strain. Then with a step-by-step approach, it will tell you how to separate the bacterial compartments according their solubility and then how to extract your protein in its native conformation using detergent. Finally, there is a detailed description on how to improve the purity of your protein via anion exchange chromatography. If you want to study bacterial porins reconstituted in their natural environment, here there is even the description on how to drive them into the outer membrane vesicles where they can be copurified. The protocol is easier and less empirical than the ones of most of the other membrane proteins and represents a solid base for soluble proteins as well (like enzyme or pore forming toxins). Expression time and temperature, concentration of the inducer, nature and concentration of the detergent, incubation time and temperature, purification pH, these are all the parameters that you can troubleshoot according to your results. The expression, extraction and purification will not last longer than a full day each and I have not yet found any protein which could not be isolated thanks to a derivation of this protocol.
