Structure and Morphology of Protein Inclusion Bodies in Escherichia Coli

Abstract

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.

Propionibacterium acnes, a resident of lipid-rich human skin, produces a 33 kDa extracellular lipase encoded by gehA

“…The overexpressed GehA polypeptide was produced as inclusion bodies which were absent from control cell extracts. The irregular shape of these inclusion bodies might support the possibility that GehA was located in the periplasm, as a previous report (Bowden et al, 1991) suggested that periplasmic inclusion bodies could be differentiated from those expressed in the cytoplasm by their irregular morphology. The formation of soluble recombinant proteins in the periplasm of E. coli is often favoured by incubation at low temperature (Schein & Noteborn, 1988;Hockney, 1994) or in the presence of non-metabolizable sugars such as sucrose (Bowden & Georgiou, 1988), and a combination of both of these incubation parameters resulted in a marked increase in the production of active lipase polypeptide.…”

Molecular Characterization of β‐Lactamase Inclusion Bodies Produced in Escherichia coli. 1. Composition

“…However, even though the conformation of the aggregated protein appears to be similar, the incluaion bodies exhibit marked differences in morphology, in their resistance to protease digestion, and in solubilization by denaturants (Bowden et al, 1991). In addition, we have found that the yield of enzymatically active @-lactamase upon GuHCl solubilization and renaturation of inclusion bodies is generally low, whereas under the same experimental conditions the purified protein can be refolded quantitatively (Valax and Georgiou, 1993).…”

“…The outcome of the in vivo folding process, i.e., whether a protein reaches its native state or forms aggregates, depends on the folding pathway, interactions with cellular components such as chaperonins, the rate of protein synthesis, and growth conditions. The formation of inclusion bodies is not necessarily undesirable and can actually offer great advantages for protein production and purification (Georgiou and Bowden, 1991). However, the recovery of active protein from inclusion bodies generally requires the complete solubilization of the aggregates under strong denaturing conditions, which usually lead to the complete unfolding of the protein.…”

“…8756-7938/93/3009-0539$04.00/0 (Bowden et al, 1991). The inclusion bodies, which became focused in a band at the interface between the 53% and 67% sucrose layers, were recovered and resuspended in water.…”

Investigation on solubilization protocols in the refolding of the thioredoxin TsnC from Xylella fastidiosa by high hydrostatic pressure approach

“…Purified recombinant IBs appear as spherical, ellipsoidal, or cylindrical particles measuring between 0.5 and 1.8 mm, characterized by a smooth and porous surface [36,37]. TsnC IBs consisted of spherical particles (Fig.…”