These include compatibility with aqueous and non-aqueous media, straightforward and cheap purification, reusability and no loss of activity due to immobilization 1.ĬatIBs are usually engineered by the addition of small peptide tags or aggregation-inducing protein domains to a protein of interest. The application of CatIBs offers several advantages compared to soluble or synthetically immobilized enzymes. As such, CatIBs can be used as immobilized enzymes, thus increasing the interest in this class of protein aggregates in recent years 1, 2, 11. coli without the need for laborious solubilization. Nonetheless, laborious downstream purification, solubilization and refolding steps are typically required to obtain the target protein in the correct (active) folding state 2, 5, 10.Ĭatalytically active inclusion bodies (CatIBs) offer a valuable alternative as they can be produced in E. For this reason, intentional aggregation of the target protein and purification of the resulting IB is a widely used strategy for product concentration and crude purification in the pharmaceutical industry. They, additionally, often contain the aggregated protein in high concentration with relatively little contamination by other intracellular proteins. IBs are currently defined as an amorphous mixture of amyloid-like cross molecular beta sheets and native (like) folded protein structures that are substantially active and could be further used as such without the need to re-fold 7, 8, 9. Additionally, the failure of the cell to post-translationally modify the protein or to form the inter- and intra-subunit disulphide bonds are reasons for IB formation 1, 2, 3, 4, 5, 6. The seed of IB formation is the presence of unproperly folded protein which is induced by several factors such as uncontrolled/unfavored growth pH and/or temperature, oxidative stresses, high rate of heterologous proteins expression (using strong expression vectors or high inducer concentrations) which exceeds the protein folding.
Heterologous expression of genes in Escherichia coli often leads to intracellular aggregation of the target overproduced protein which are called inclusion bodies (IBs). These results show that the recombinant thermostable TkAMPpase is one of rare examples of naturally formed CatIBs. Interestingly, differences in the substrate preference were observed. coli were found to be similarly active with 2–6 folds higher specific activity compared to these heat-induced aggregates. Additionally, inclusion bodies formed during the expression in E. Herein, we show that heat treatment of soluble protein from crude extract induces aggregation of active protein which phosphorolyse all natural 5′-mononucleotides. Previous studies have revealed that the adenosine 5′-monophosphate phosphorylase of Thermococcus kodakarensis ( TkAMPpase) forms large soluble multimers with high thermal stability. However, very few examples of CatIBs that are naturally formed during heterologous expression have been reported so far. They can be purified easily and used directly as stable and reusable heterogenous catalysts. Catalytically active inclusion bodies (CatIBs) produced in Escherichia coli are an interesting but currently underexplored strategy for enzyme immobilization.
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