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

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Solubilization of Expressed Proteins from Inclusion Bodies

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The expression of foreign proteins at high levels in E. coli often results in the formation of cytoplasmic granules or inclusion bodies composed of insoluble aggregates of the expressed protein. These inclusion bodies can be seen with a phase-contrast microscope and are readily separated from most soluble and membrane-bound bacterial proteins, as described in this protocol. Briefly, cells expressing high levels of foreign protein are harvested by centrifugation and lysed by mechanical techniques, sonication, or lysozyme plus detergents. As with all lysis procedures, it is crucial to obtain maximum cell lysis to obtain inclusion bodies in high yields. The inclusion bodies have a high density and are recovered by centrifugation (insoluble pellets) and washed. The purpose of the washing steps is to remove as much soluble, adherent bacterial protein as possible from the aggregated foreign protein. In most cases, adjusting the washing conditions allows isolation of inclusion bodies that contain >90 pure foreign protein. The washed inclusion bodies are then solubilized in detergents or denaturants, and the denatured target protein is refolded by gradually removing the denaturant. Various denaturants (e.g., guanidine HCl [58 M], urea [68 M], SDS, alkaline pH, or acetonitrile/propanol) may be used to solubilize the inclusion bodies. Each protein may require a slightly different procedure, which must be determined empirically (see, e.g., Patra et al. 2000; Tan et al. 2007). The procedures given below use washing with Triton X-100 and EDTA (Marston et al. 1985; Estap and Rinas 1996) or urea (Schoner et al. 1992) followed by solubilization in high concentrations of urea (8 M) and alkaline treatment (pH 10.7). Triton X-100 and EDTA have been used to solubilize prorenin inclusion bodies (Marston et al. 1985). The material extracted from the purified inclusion body can be used directly as an antigen (Harlow and Lane 1988). Alternatively, refolding techniques can be used to attempt to recover active protein. The probability of successfully refolding the proteins isolated from inclusion bodies into soluble and functionally active forms is unfortunately low (Cabrita and Bottomley 2004; Graslund et al. 2008). The production of soluble proteins is almost always preferred over refolding from inclusion bodies and should be attempted first. For details on expression optimization that can reduce the production of inclusion bodies, see Protocol 1. Nevertheless, the potential for high yield and purity may make a refolding effort worthwhile for small intracellular proteins before turning to alternative expression systems. Several options for refolding the solubilized proteins are outlined in the box Considerations for Refolding Solubilized Proteins Recovered from Inclusion Bodies.

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