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

Abstract

We have determined the macromolecular composition of inclusion bodies formed by overexpressing P-lactamase from three different expression systems as a function of the growth conditions. The inclusion bodies were purified by differential gradient centrifugation and detergent extraction. Both the expression system and the growth conditions were shown to have a pronounced effect on inclusion body composition.Specifically, contaminating polypeptides ranged from less than 5 % to over 50% of the total protein content. Phospholipids composed 0.5-13 % of the inclusion bodies. Nucleic acids represented a minor impurity for both cytoplasmic and periplasmic inclusion bodies. Cytoplasmic inclusion bodies of the mature P-lactamase had the lowest amount of impurities, irrespective of the growth conditions. On the other hand, large amounts of outer membrane proteins and phospholipids were observed in periplasmic inclusion bodies from cells grown a t basic pH. Our results show that, at least under some growth conditions, protein aggregation i n vivo is highly specific, and the presence of contaminating proteins in inclusion bodies is due to incomplete purification following cell lysis.

Depletion of the Signal Recognition Particle Receptor Inactivates Ribosomes in Escherichia coli

“…3B). Inclusion bodies are normally removed by the first centrifugation that results in the S30 extract (6,40,47); the detection of IbpA/IbpB in the pellet fraction of the S135 extract ultracentrifugation product, however, suggests that not all protein aggregates/inclusion bodies are removed by this first centrifugation. The formation of inclusion bodies could possibly explain the altered sedimentation behavior of ribosomes (c.f.…”

High pressure fosters protein refolding from aggregates at high concentrations

“…These structures, referred to as inclusion bodies, contain a high level of non-native intermolecular ␤-sheet structure and pose a serious obstacle to efficient production of recombinant proteins. To test the applicability of high pressures to obtaining native proteins from inclusion bodies, we studied a cytoplasmic inclusion body system in E. coli (15) that contains ␤-lactamase as a major protein component. IR spectroscopy of harvested and washed (15) ␤-lactamase inclusion bodies documented that this system contains a large fraction of non-native intermolecular ␤-sheet structure (data not shown).…”

“…Once soluble and unfolded, the proteins are first diluted with additional GdmHCl solution and then refolded by removing the chaotrope by dialysis or additional dilution. The refolding step, however, is difficult and depends strongly on renaturing conditions (14,15). For example, redox conditions, pH, rates of dialysis, and protein concentration all must be empirically optimized for each protein (16).…”

Folding and aggregation of TEM β‐lactamase: Analogies with the formation of inclusion bodies in Escherichia coli

“…The cultures were induced with M isopropyl-@-D-thiogalactoside at an OD, of about 0.35. Under these conditions, 0-lactamase accumulates in soluble form at a level exceeding 30% of the total cell protein and no accumulation of unprocessed pre-@-lactamase precursor is observed (Valax & Georgiou, 1993). After overnight growth, the cells were harvested by centrifugation at 10,000 X g for 10 min, resuspended in lOmM Tris acetate buffer, pH 8.0, containing 0.75 M sucrose, and then converted to spheroplasts according to the procedure described by Osborn and Munson (1976).…”