- Poster Presentation
- Open Access
Comparative analysis of E. coli inclusion bodies and thermal protein aggregates
© González-Montalban et al; licensee BioMed Central Ltd. 2006
- Published: 10 October 2006
- Inclusion Body
- Thermal Denaturation
- Protein Deposition
- Protein Quality Control
- Heat Shock Stress
In bacteria, inclusion bodies are commonly observed during overexpression of plasmid-encoded recombinant genes, and represent a great matter of concern in biotechnology . Bacterial inclusion bodies are also connected to the protein quality control  and to the prevention of cytotoxicity associated to aberrantly folded proteins [3, 4]. On the other hand, these protein aggregates are dynamic structures, since they grow as the result of an unbalanced equilibrium between protein deposition and removal [5, 2]. Therefore, there is not any physiological evidence of bacterial inclusion bodies being structures well organized to facilitate embedded protein removal by chaperones or proteases. We have comparatively analyzed the molecular organization and dynamism of a recombinant E.coli β-galactosidase and its derivative VP1LAC  when either deposited as inclusion bodies or as aggregates resulting from in vivo thermal denaturation in a laboratory wild type strain E.coli MC4100 and its derivatives DnaK- and GroEL44 (namely JGT20 and BB4565, respectively). The expression of both lacZ and VP1LAC genes is triggered by temperature up shift from 28°C to 42°C.
Specific activity (in U/ng) of β-galactosidase and its derivative VP1LAC produced in different strains, in the soluble and insoluble fractions.
628.2 ± 40.5
6.3 ± 0,3
234.1 ± 52.9
65.2 ± 19,4
689.7 ± 164.9
63.6 ± 2,2
230.2 ± 25.7
129.6 ± 45,9
888.9 ± 179.3
175.2 ± 34,9
12.5 ± 3.8
10.3 ± 6.3
Thermal denaturation of β-galactosidase results in the formation of heterogeneous aggregates that are rather stable in composition during the heat shock stress. On the contrary, protein deposition as inclusion bodies renders homogeneous but strongly evolving structures. In this context, the specific activity of enzyme-based inclusion bodies is much higher than in the equivalent thermal aggregates, by a mechanism that might be controlled by the chaperone DnaK. Protein deposition as inclusion bodies is then a cell driven complex process through which misfolded protein forms but also functionally competent polypeptides are efficiently packaged.
This work has been funded by BIO2004-00700 from MEC, Spain and 2005SGR-00956 (AGAUR). Núria González-Montalbán and Elena García-Fruitós are recipients of doctoral fellowships from MEC, Spain, and Salvador Ventura is supported by a "Ramón y Cajal" project awarded by the MCYT an co-financed by the Universitat Autònoma de Barcelona.
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