Isolating inclusion bodies from bacteria.
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Bacteriolysis↗
Biomedical subjects
P Valax
Publications and source records attributed to P Valax.
Expression of correctly folded proteins in Escherichia coli.
Many heterologous polypeptides fail to fold into their native state when expressed in Escherichia coli; instead, they are either degraded by the cellular proteolytic machinery or accumulate in insoluble form, typically as inclusion bodies. Misfolding is a particularly vexing problem in the expression of mammalian proteins, especially those that are composed of multiple subunits, have several disulfide bonds, or contain prosthetic groups. Fortunately, bacteria exhibit a remarkable physiological plasticity that can be successfully exploited to improve protein folding. Significant yields of active heterologous proteins have been obtained through strategies that include the co-expression of homologous or heterologous folding accessory proteins, the optimization of growth conditions, and the use of fusion proteins. A flood of recent reports documenting the successful production of complex eukaryotic proteins in active form have demonstrated that bacteria can provide the proper environment for the folding of the vast majority of recombinant polypeptides.
Bacterial Proteins↗
Folding and aggregation of TEM beta-lactamase: analogies with the formation of inclusion bodies in Escherichia coli.
The enzyme TEM beta-lactamase has been used as a model for understanding the pathway leading to formation of inclusion bodies in Escherichia coli. The equilibrium denaturation of TEM beta-lactamase revealed that an intermediate that has lost enzymatic activity, native protein fluorescence, and UV absorption, but retains 60% of the native circular dichroism signal, becomes populated at intermediate (1.0-1.4 M) concentrations of guanidium chloride (GdmCl). This species exhibits a large increase in bis-1-anilino-8-naphthalene sulfonic acid fluorescence, indicating the presence of exposed hydrophobic surfaces. When TEM beta-lactamase was unfolded in different initial concentrations of GdmCl and refolded to the same final conditions by dialysis a distinct minimum in the yield of active protein was observed for initial concentrations of GdmCl in the 1.0-1.5 M range. It was shown that the lower reactivation yield was solely due to the formation of noncovalently linked aggregates. We propose that the aggregation of TEM beta-lactamase involves the association of a compact state having partially exposed hydrophobic surfaces. This hypothesis is consistent with our recent findings that TEM beta-lactamase inclusion bodies contains extensive secondary structure (Przybycien TM, Dunn JP, Valax P, Georgiou G, 1994, Protein Eng 7:131-136). Finally, we have also shown that protein aggregation was enhanced at higher temperatures and in the presence of 5 mM dithiothreitol and was inhibited by the addition of sucrose. These conditions exert a similar effect on the formation of inclusion bodies in vivo.
Anilino Naphthalenesulfonates↗
Secondary structure characterization of beta-lactamase inclusion bodies.
The secondary structure of proteins in E. coli inclusion bodies was investigated via Raman spectroscopy. Inclusion bodies were purified from cells expressing different forms of RTEM beta-lactamase and grown at either 37 or 42 degrees C. All of the solid phase inclusion body samples examined gave amide I band spectra that were perturbed from that of the native, purified protein in both solution and powder forms; secondary structure estimates indicated significant decreases in alpha-helix and increases in beta-sheet contents in the inclusion body samples. The structure estimates for inclusion bodies isolated from 37 degrees C cultures were similar, regardless of aggregate localization in the E. coli cytoplasmic or periplasmic spaces or beta-lactamase precursor content. Inclusion bodies obtained from 42 degrees C cells exhibited a further reduction of alpha-helix and augmentation of beta-sheet contents relative to those from 37 degrees C cultures. These results are consistent with the paradigm for inclusion body formation via the self-association of intra-cellular folding intermediates having extensive secondary structure content. Further, the overall secondary structure content of inclusion bodies is not significantly affected by subcellular compartmentalization, but may be altered at increased temperatures.
Escherichia coli↗
Molecular characterization of beta-lactamase inclusion bodies produced in Escherichia coli. 1. Composition.
We have determined the macromolecular composition of inclusion bodies formed by overexpressing beta-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 beta-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 at basic pH. Our results show that, at least under some growth conditions, protein aggregation in vivo is highly specific, and the presence of contaminating proteins in inclusion bodies is due to incomplete purification following cell lysis.
Bacterial Proteins↗