Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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Preparation of Cell Extract for Purification of Soluble Proteins Expressed in E. coli

(Protocol summary only for purposes of this preview site)

Recovery of intracellular proteins requires disruption of the host cell before the target protein is extracted and isolated. Disruption methods vary depending on the type of cells, the total volume, and the number of samples being processed. The methods commonly used include, but are not limited to, hypotonic shock, enzymatic lysis by lysozyme, sonication, and microfluidic or French press mechanical disruption (for review, see Grabski 2009). In some cases, cells may be easily lysed using a hypotonic shock method wherein the cells are exposed to a hypotonic solution and lysed by the osmotic pressure that results from rapid uptake of water. For cells enveloped in cell walls (such as E. coli and yeast), mild techniques such as hypotonic shock are not sufficient to achieve adequate lysis. More vigorous methods, such as sonication or the use of a French press, are often required. Although these forceful methods are more effective in cell wall lysis, they also increase the risk of damaging the target protein with local heating, mechanical shear, oxidation, and free radical reactions (Hawkins and Davies 2001; Mason and Peters 2002). For this reason, it is often essential to optimize the lysis protocol for each cell line and technique to maximize the activity and quantity of recovered protein, while minimizing the time and instrumentation required (Benov and Al-Ibraheem 2002).


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(Limited time special offer.) Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook
Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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