Abstract
BackgroundIn recent years bacterial inclusion bodies (IBs) were recognised as highly pure deposits of active proteins inside bacterial cells. Such active nanoparticles are very interesting for further downstream protein isolation, as well as for many other applications in nanomedicine, cosmetic, chemical and pharmaceutical industry.To prepare large quantities of a high quality product, the whole bioprocess has to be optimised. This includes not only the cultivation of the bacterial culture, but also the isolation step itself, which can be of critical importance for the production process.To determine the most appropriate method for the isolation of biologically active nanoparticles, three methods for bacterial cell disruption were analyzed.ResultsIn this study, enzymatic lysis and two mechanical methods, high-pressure homogenization and sonication, were compared.During enzymatic lysis the enzyme lysozyme was found to attach to the surface of IBs, and it could not be removed by simple washing. As this represents an additional impurity in the engineered nanoparticles, we concluded that enzymatic lysis is not the most suitable method for IBs isolation.During sonication proteins are released (lost) from the surface of IBs and thus the surface of IBs appears more porous when compared to the other two methods. We also found that the acoustic output power needed to isolate the IBs from bacterial cells actually damages proteins structures, thereby causing a reduction in biological activity.High-pressure homogenization also caused some damage to IBs, however the protein loss from the IBs was negligible. Furthermore, homogenization had no side-effects on protein biological activity.ConclusionsThe study shows that among the three methods tested, homogenization is the most appropriate method for the isolation of active nanoparticles from bacterial cells.
Highlights
In recent years bacterial inclusion bodies (IBs) were recognised as highly pure deposits of active proteins inside bacterial cells
Results show that non-classical IBs (ncIBs) can be isolated from E. coli using only enzymatic lysis, but some impurities still remain in the sample and cannot be washed off by simple washing (Figure 1, 3, 4 and 5)
Enzymatic lysis was determined as the method that is most gentle towards ncIBs since no mechanical forces that would damage the surface of ncIBs or the biological activity of the target protein inside ncIBs, are employed
Summary
In recent years bacterial inclusion bodies (IBs) were recognised as highly pure deposits of active proteins inside bacterial cells. Such active nanoparticles are very interesting for further downstream protein isolation, as well as for many other applications in nanomedicine, cosmetic, chemical and pharmaceutical industry. The rapid expansion of biotechnology has lead to the production of a wide spectrum of recombinant proteins. To this end, a range of host organisms, from bacteria to mammalian cell-culture systems are being used. IBs possessing active proteins can be used as active protein nanoparticles, with many possible applications [5,6,13,14]
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