We have studied the structure and characteristics of inclusion bodies formed by the enzyme beta-lactamase in the periplasmic space of Escherichia coli or in the cytoplasm, following expression of the protein without its signal sequence. Electron microscopy of highly purified protein aggregates using a novel sucrose gradient centrifugation procedure revealed striking morphological differences. Periplasmic inclusion bodies were essentially amorphous whereas the protein particles in the cytoplasm were highly regular. Depending on the cellular location, the inclusion bodies exhibited differences in protein composition even though they were formed by the expression of the same polypeptide chain. It was shown that the chaperonins GroEL and SecB are not incorporated into the inclusion bodies. Furthermore, the degree of solubilization of the inclusion bodies in the presence of denaturants and the sensitivity of the aggregated proteins to protease digestion indicated that the differences between cytoplasmic and periplasmic inclusion bodies extend to the conformation of the associated polypeptide chains.

13-Lactamase with the -20 to -1 region of the leader peptide deleted (almost complete deletion of the leader peptide) [A(-20,-1) 13-lactamasel was released from Escherichia coli cells by osmotic shock. Fractionation of the cells by conversion to spheroplasts and protease accessibility experiments further indicated that a portion of the protein may be bound to the cytoplasmic membrane and be partially exposed in the periplasmic space.Expression of A(-20,-1) ,-lactamase conferred a 25-fold increase in the 50%o lethal dose for ampicillin relative to that for controls, thus confirming that a small amount (about 2%) of the active protein is completely exported from the cytoplasm. These results suggest that even in the absence of a leader peptide, mature 13-lactamase is able to interact with the cytoplasmic membrane and be translocated into the periplasmic space, albeit with a low efficiency.Essentially all exported proteins in Escherichia coli are synthesized as precursors containing a leader peptide (22,25,28). The most important function of the leader peptide is probably to maintain the precursor in an export-competent conformation, a process which is facilitated by interactions with chaperonins. According to the kinetic competition model suggested by Pugsley (20) and elaborated by Hardy and Randall (10), protein precursors destined for secretion are able to bind to chaperonins, such as GroEL and SecB, partly because they exhibit slower folding kinetics. Cytoplasmic proteins, on the other hand, reach their native state much more rapidly and do not have an opportunity to enter the export pathway.Certain mature proteins expressed without a leader peptide and a few bona fide cytoplasmic polypeptides are found together with periplasmic components following cell fractionation by osmotic shock. Proteins which appear in the osmotic shock fraction in the absence of a leader peptide include Vitreoscilla hemoglobin, human proapolipoprotein, and mouse interleukin-13 expressed in E. coli and the native cytoplasmic proteins thioredoxin and elongation factor Tu (2,11,13,23). Even though cell fractionation techniques, particularly osmotic shock, are prone to artifacts (18), it is conceivable that the unexpected fractionation behavior of the above proteins stems from their atypical localization within the cell. For example, there is evidence that thioredoxin is found predominantly in adhesion sites between the inner and outer membranes (2).In this report, we show that P-lactamase expressed without a functional leader peptide is found associated with the cytoplasmic membrane and the periplasmic fraction, provided that the cells are grown under conditions which prevent "trapping" of the protein within cytoplasmic inclusion bod- 3-Lactamase in the osmotic shock fraction and in the lysed cells was measured as described previously (24). The sum of activities obtained after cell fractionation agreed, within 10%, with the values determined from assays of total-cell lysates. In all experiments, the release of ,B-galactosidase activity (...

Overproduction of the secreted protein p-lactamase in Escherichia coli results in the formation of inclusion bodies in the periplasmic space. The aggregation of p-lactamase was inhibited when non-metabolizable sugars, such as sucrose and rafinose, were added into the growth medium. A four-fold increase in the amount of solubleprotein was obtained under optimal conditions. The mechanism for the inhibition of protein aggregation was investigated. Addition of 0.3M sucrose decreased the rate of processing of the p-lactamase precursor, causing its transient accumulation within the cell.determined using the Bio-rad assay with Bovine Serum Albumin as the standard. SDS-PAGE was performed in 15% gels [9]. Proteins were concentrated by lyophilization prior to electrophoresis. Results and DiscussionThe inducible tac promoter has been used to express p-l'actamase to a level corresponding to 30% of the total soluble protein [9]. In uninduced cultures, p-lactamase is secreted

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