Embedded Files
Batch dilution refolding harnessing recyclable molecular chaperone systems
Thrombin-like-enzymes (TLEs) have long been regarded as potentially useful therapeutic agents for the treatment of blood clotting disorders through their anti-coagulant action. Gloshedobin, recently isolated from the snake venom of Gloydius shedaoensis, was demonstrated to exhibit TLE activities. Due to the difficulties incurred with its separation and purification along with the limited supply of the natural snake venom, it is difficult to obtain a sufficient amount of the enzymes for both research and clinical purposes. In general, Escherichia coli offers a route for the rapid and economical production of recombinant proteins. However, over-expression of recombinant gloshedobin in E. coli led to intracellular accumulation as solid aggregates known as inclusion bodies (IBs), which requires solubilization and subsequent refolding to regain biological activity. In this project, we would like to develop novel refolding cocktail based refolding strategies, departing from the conventional refolding methods reported elsewhere, by harnessing in vitro realization of a molecular chaperone network for renaturation of solubilized but denatured gloshedobin in a manner to mimic cellular folding machinery. In order to redesign the traditional batch dilution refolding schemes, molecular chaperones (e.g. DnaKJE & ClpB) are coupled with two types of beads: 1) in-house cloned His-tagged molecular chaperones immobilized on the metal chelating beads, and 2) chaperones covalently bound to the CNBr-activated Sepharose 4B. The important advantage of the proposed refolding method is that separation between chaperones and target protein can be facilitated in the post-refolding purification steps, thereby allowing the repeated use of chaperone-loaded beads for next refolding reactions with an expectation of significantly improved process economics. Furthermore, this approach will realize the folding-like-refolding strategy by allowing coupling/decoupling of protein substrates at high concentrations with chaperone-loaded beads in the presence of ATP regeneration system, which closely mimics the bacterial way of conducting protein quality control.
Chaotrope-free folding-like-refoldingof IB proteins
Escherichia coli has been one of the popular biofactories for the production of recombinant proteins due to high production rate, simple expression system and low production cost. However, heterologous protein expression in E. coli often leads to formation of insoluble inclusion bodies (IBs), which entails the complicated processing steps such as IBs extraction, IBs solubilization and refolding of denatured IB proteins. Among these, refolding is the most challenging step because it is a bottleneck to determine the success of overall IBs processing, accounting for more than 70% of the overall processing cost. The most conventional industrial refolding process is based on batch dilution refolding where a large volume of cost-prohibitive refolding buffer hence a large refolding vessel and extended processing time are typically required to minimize protein aggregation during refolding reaction. We have demonstrated that a novel refolding strategy (i.e. folding-like-refolding) harnessing a refolding cocktail which comprises molecular or artificial chaperones and ATP regeneration system could radically enhance the efficiency of refolding for both heat-denatured malate dehydrogenase (MDH) and urea-denatured gloshedobin following its IB-route expression and solubilization. To the best of our knowledge, all the reported refolding schemes by far requires solubilization of IBs prior to attempting refolding, which clearly departs from the bacterial way of processing nascent polypeptides, folding intermediates, and/or aggregated proteins. For a full realization of “folding-like-refolding” concept, this project aims to develop a new refolding strategy capable of achieving in situ chaotrope-free synchronized solubilization and refolding of cytoplasmic IBs by precisely mimicking cellular disaggregation and folding mechanisms with the use of optimized refolding cocktail and redox environment.
Harnessing molecular chaperone refolding cocktail to develop folding-like-refolding strategy: Dependency of chaperoning effect on the size of protein aggregates
Proteins have wide applications in the medical, industrial and agricultural fields. They are usually produced in huge quantities in host cells such as Escherichia coli. However, high-level expression of recombinant proteins in E. coli often results in accumulation of insoluble aggregates known as inclusion bodies (IBs), thus requiring further solubilization, refolding and purification procedures to achieve functionally active products. Molecular chaperones have been applied successfully to refold various proteins both in vivo and in vitro, opening a new era in protein refolding. However, the exact function of individual molecular chaperones and the interaction between target protein and molecular chaperones are still unclear. We demonstrated that refolding cocktail comprising ClpB/DnaKJE with or without the presence of ATP regeneration system and a refolding additive, PEG, could significantly enhance the refolding efficiency of heat-denatured malate dehyrogenase (MDH) and chaotrope-solubilized gloshedobin following its expression as IBs. To further clarify the individual or synergistic roles of each chaperone, various recombinant molecular chaperones including His-ClpB, His-DnaK, His-DnaJ, His-GrpE, His-Gro EL, His-Gro ES, and His-Trigger Factor were recently cloned, expressed and purified through IMAC, respectively. Currently, chaperoning efficiencies of these His-tagged molecular chaperones are being studied systematically using heat- or chemical-denatured model proteins of different aggregate size. It is expected that our study will provide a better understanding of chaperone-assisted refolding process, thereby facilitating engineering implementation of a novel refolding strategy, folding-like-refolding approach based on refolding cocktail.
Page updated
Google Sites
Report abuse