Abstract
BackgroundBacterial inclusion bodies are submicron protein clusters usually found in recombinant bacteria that have been traditionally considered as undesirable products from protein production processes. However, being fully biocompatible, they have been recently characterized as nanoparticulate inert materials useful as scaffolds for tissue engineering, with potentially wider applicability in biomedicine and material sciences. Current protocols for inclusion body isolation from Escherichia coli usually offer between 95 to 99% of protein recovery, what in practical terms, might imply extensive bacterial cell contamination, not compatible with the use of inclusion bodies in biological interfaces.ResultsUsing an appropriate combination of chemical and mechanical cell disruption methods we have established a convenient procedure for the recovery of bacterial inclusion bodies with undetectable levels of viable cell contamination, below 10-1 cfu/ml, keeping the particulate organization of these aggregates regarding size and protein folding features.ConclusionsThe application of the developed protocol allows obtaining bacterial free inclusion bodies suitable for use in mammalian cell cultures and other biological interfaces.
Highlights
Bacterial inclusion bodies are submicron protein clusters usually found in recombinant bacteria that have been traditionally considered as undesirable products from protein production processes
To explore the efficiency of conventional inclusion bodies (IBs) purification protocols based on lysozyme treatment combined with repeated detergent washing steps, we have determined the number of viable bacterial cells before and after cell lysis, using different bacterial strains (MC4100, DnaKand ClpP-) carrying plasmids encoding different recombinant proteins (VP1GFP, VP1LAC and VP1NLSCt) (Figure 1)
Our results indicate that the used standard protocol is inefficient concerning the complete removal of viable bacteria (Figure 1)
Summary
Bacterial inclusion bodies are submicron protein clusters usually found in recombinant bacteria that have been traditionally considered as undesirable products from protein production processes. Bacterial inclusion bodies (IBs) are water-insoluble protein aggregates formed in the bacterial cytoplasm (and eventually periplasm) during the overproduction of recombinant proteins, especially those from viral or mammalian origin [1]. IBs are pure [8], structurally organized [9], mechanically stable and biocompatible protein deposits [2], formed through stereospecific protein-protein cross-molecular interactions under amyloidlike schemes [9,10] This turn in the understanding of IB biological nature has prompted to explore potential. Bacterial lysis is achieved using either mechanical or non mechanical methods, or a combination of both, while washing steps include, among others, detergent and/or DNase treatments These protocols have been mainly aimed to obtain IBs suitable for in vitro protein refolding attempts [15]. For applications in which IBs act as materials in biological interfaces, for instance in tissue engineering [2] or as biocatalysts [11], the presence of living bacteria would be not acceptable
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