Localizing Protein

When proteins incorporating a secretion vector are expressed in E. coli, advantage can be taken of the fact that the recombinant proteins will be located in the periplasmic space and/or the culture medium. Secretion into the medium is due to "leakage" from the periplasm and appears to depend on the level of accumulation and the fermentation conditions. Figure 6.1.2 summarizes approaches used to recover proteins selectively from the periplasmic space or the medium (see also unit 5.2). High-level secretion into the periplasm sometimes results in the formation of aggregates, analogous to cytoplasmic inclusion bodies (Bowden et al., 1991). Periplasmic inclusion bodies can be extracted from the low-speed pellet fraction following normal cell breakage (see Fig. 6.1.1).

Proteins in the medium can be recovered by subjecting the culture medium to centrifu-gation or filtration, steps that remove intact cells and large debris. The clarified protein is usually dilute and is often concentrated prior to purification by affinity or conventional chromatography. Periplasmic proteins can be selectively released by osmotic shock (preferred method) or by selective disruption of the outer membrane and peptidoglycan layer using lysozyme.

Apart from its use in dissecting the bacterial compartments, lysozyme is often employed to prepare complete cell lysates, especially in laboratories that do not have access to a French press. Cells treated with lysozyme can be disrupted with detergents or by brief sonication (unit 6.5).

Useful microscale (<1 ml) E. coli cell fractionation schemes have been based on osmotic shock treatment (Yarranton and Mountain, 1992) or repeated freezing and thawing of cells (Johnson and Hecht, 1994). unit 5.2 describes small-scale (1- to 25-ml) procedures for preparing samples of periplasmic extracts and extracellular media for analysis by SDS-PAGE ( unit 10.1).

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